Introduction: Cardiac troponin is one of the heart biomarkers and its high levels correlates with a high risk of cardiomyocytes damage. This study aimed to compare sevoflurane and isoflurane effect on troponin levels in patients undergoing cardiac surgery.  Methods:  We systematically searched for RCTs which had been published in Cochrane library, PubMed, Web of science, CRD, Scopus, and Google Scholar by the end of February 30th, 2019. The quality of articles was evaluated with the Cochrane checklist. GRADE was used for quality of evidence for this meta-analysis. Meta-analysis was done based on random or fixed effect model. Results: Five studies with total of 190 (sevoflurane) and 191 (isoflurane) patients were included. The results showed that pooled mean difference of troponin levels between the two groups was significant at ICU admission time and 24 hours after entering. The comparison of troponin level changes between the two groups (baseline = at time ICU) in 24 and 48 hours after ICU admission was significant. Conclusion: This meta-analysis showed that blood troponin levels were significantly lower at the time of arrival in ICU with isoflurane and after 24 hours with sevoflurane. Generally, given the small mean difference between isoflurane and sevoflurane, it seems that none of the medications has a negative effect on the cardiac troponin level. Cardiac enzymes such as troponin T and I are proprietary biomarkers which are not detectable in healthy people.6 Troponins are regulatory proteins present in actin filaments that contribute to the regulation of the contraction of the heart cells. When a patient suffers from heart attack, the amount of these enzymes changes and increases. Therefore, troponins are considered as high sensitivity markers to measure patients’ cardiac status.9Cardiovascular disease is the most common cause of death. One of the approaches considered for the treatment of patients with this disease is cardiac surgery. Patients undergoing heart surgery are usually at risk of developing myocardial damage.11 These two volatile anesthetics have different effects on cardiac function.12 However, sevoflurane is less soluble than isoflurane.13 Several animal studies have shown that volatiles anesthesia has a protective effect on myocardial infarction.17 Human studies also indicate that these drugs have the potential to provide adequate anesthetic depth. Moreover, these drugs have protective effects on myocardial infarction. Furthermore, research studies, which have examined the amount of troponin enzyme using isoflurane and sevoflurane judges, have shown different results.21 Troponin often presents a unique prognosis of patient conditions in the ICU and 24 hours after the operation.22Today, the use of volatiles anesthetics such as isoflurane and sevoflurane has expanded in many surgical procedures, including heart surgery.In this regard, the aim of this study was to compare the effect of isoflurane and sevoflurane on troponin levels in patients undergoing cardiac surgery in a systematic review and meta-analysis. The results of this study would help clinicians obtain evidence based on clinical evidence and select the best inhalational anesthetic agent.23This was a systematic review and meta-analysis study that utilized randomized controlled trials (RCTs) to compare the effects of sevoflurane and isoflurane on troponin levels in patients with cardiac surgery, without any time constraints and based on Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines.We systematically searched for RCTs published in Cochrane library, PubMed, Web of science, CRD, Scopus and Google Scholar databases until February 30, 2019. Keywords include ‘Sevoflurane’, ‘Isoflurane’, ‘Anesthesia’, Coronary artery bypass surgery’, ‘Heart surgery’, ‘randomized controlled trials’, and corresponding MeSH terms. The reference list of selected studies was searched manually. Hand-searching and studies presented at conferences were also searched  they studied patients who underwent cardiac surgery and reported the results; (2) patients had to have at least one troponin measurement during cardiac surgery; (3) patients undergoing cardiac surgery used sevoflurane and isoflurane in their anesthesia; and (4) the study was a prospective, randomized, controlled clinical trial. Exclusion criteria were (1) comparison of other anesthetic drugs; (2) other kinds of surgeries; (3) studies that did not report on specific outcomes; (4) studies that had incomplete data about the mean and standard deviation of the desired outcomes; and (5) animal studies.The Endnote X5 resource management software was used to organize, study the titles and abstracts, and identify duplicates. After removing duplicate articles, the abstracts and the full texts of the articles were examined and those which met eligibility criteria were selected. The extracted data were transferred into a table. The extracted data include: First author, year of publication, the country in which the study had been performed, type of interventions, the number of subjects in the control and intervention group, type of study, mean and standard deviation of troponin levels, and finally  random sequence generation (selection bias), (ii) allocation concealment (selection bias), (iii) blinding of participants and personnel (performance bias), (iv)blinding of outcome assessment (detection bias), (v) incomplete outcome data (attrition bias), (vii) selective reporting (reporting bias), and other bias.The quality evaluation of the studies was done using the Cochrane Collaboration’s toolDuring all stages of study selection, data extraction and quality assessment of studies were done by two of the researchers independently. Any disagreement between the researchers was discussed with a third reviewer and the consensus was gained25 In the present study, this approach measured the strength of the evidence obtained from primary and secondary outcomes as well as subgroups from low to high in order to study the strength of GRADE evidence, heterogeneity between studies was considered by Indirectness, Imprecision, Publication bias and Power of relation.A structured and transparent GRADE approach study was performed .2 statistics which expresses the percentage of variation between studies. I2 values less than 25% indicated low heterogeneity, I2 values between 25% and 75% was regarded average heterogeneity, and over 75%, heterogeneity was considered high. In case of significant heterogeneity, the random effects model was used to calculate the overall effect size. Statistical analysis was performed using CMA v.2.0 software and P value less than 5% was considered as a significant level.The sample size, mean troponin level, and standard deviation were extracted from the studies before the induction time, at the time of being in ICU, and 6, 12, 24 and 48 hours after entering the ICU. The difference in mean troponin levels at the above mentioned time points was calculated in both groups of sevoflurane and isoflurane compared to before the induction levels. Finally, using meta-analysis based on random or fixed effect model, the mean differences in troponin levels in the anesthetics group of sevoflurane and isoflurane were combined. The heterogeneity between studies was investigated using chi-square q-test and IP value <0.1 was considered as a sign of publication bias. In the Egger’s regression test, the mean changes in troponin levels in the two groups (sevoflurane and isoflurane) were considered 24 hours after entering the ICU.To test the Publication bias, Egger’s regression test was used. Moreover, In the studies included in this meta-analysis, the troponin level in types I and T was reported. Sub-groups were performed based on the type of troponin in both groups of sevoflurane and isoflurane in patients undergoing cardiac surgery. Sensitivity analysis was performed by removing the studies in which the level of troponin in types I and T was reported in children.27 The PRISMA flow diagram for clinical trials is shown in In the systematic search performed in databases, 4153 articles were identified. 1431 duplicate articles were recorded, and 2704 articles were excluded after reviewing their titles and abstracts. After reviewing the full texts of the articles, 13papers were excluded from the study. Finally, 5 eligible studies were included for the meta-analysis.11 and four other studies had been conducted on adults.27 The mean age of children in the sevoflurane and isoflurane groups were 3.07 and 3.47 respectively. Moreover, the mean age of adults in sevoflurane and isoflurane groups were 60.16 and 59.89 respectively. All studies assessed troponin within 48 hours after surgery. Furthermore, two studies measured cardiac troponin cTnT27 and three studies measured cardiac troponin cTnI.20 The characteristics of the included studies are shown in The final 5 selected studies had been published between 2007 and 2017. A total of 381 participants in our meta-analysis (190 in the sevoflurane group and 191 in the isoflurane group) were anesthetized. One study had been conducted on childrenIn this study, changes in cardiac troponin levels isoflurane and sevoflurane anesthesia was investigated 7 times. Form evidence obtained from the meta-analysis, this is the grading place during these seven time periods. Based on the GRADE approach, the power of evidence were in a low level at the time of ICU, 6, 12, 48 hours and (Baseline=At time 24 h (ICU)), in moderate level at the time 48 ours , and in high level at the time 24 ours. The result of GRAD is shown in 26 In one of the studies , whilst there was no explanation for the Attrition sample, there might have been an attrition bias.12 Only one study had sufficient explanations about blinding outcome assessor and the rest of the studies might have been affected by the detection bias20 . Based on meta-analysis, troponin levels in patients with anesthesia group of isoflurane were 3.63 ng/mL less than those in the sevoflurane anesthetic group. This difference was statistically significant. . A forest plot is available in Three studies reported troponin levels at the time of admission to the ICU. Two of studies had reported the cardiac troponin I and one of the studies had reported the cardiac troponin T. The homogeneity between the studies was significant . A forest plot is available in Three studies reported cardiac troponin6 hours after entering the ICU. In these studies, cardiac troponin I had been investigated. The heterogeneity among these studies was not significant . Based on meta-analysis, troponin levels were 0.36 ng/mL less in patients anesthetized with anesthetized isoflurane than those given sevoflurane with anesthesia. This difference was not statistically significant . A forest plot is available in Two studies reported cardiac troponin 12 hours after entering the ICU. In these studies, cardiac troponin I was studied. This difference was statistically significant . A forest plot is available in Four studies reported cardiac troponin levels within 24 hours of entering the ICU. In these studies, 3 studies about cardiac troponin I were investigated, and the difference was statistically significant Q=13.73; df = 1; P < 0.001; I2 = 97.44). Based on meta-analysis, cardiac troponin levels were 0.36 ng/mL more in patients treated with anesthetized isoflurane than those given sevoflurane with anesthesia. This difference was not statistically significant . A forest plot is available in Two studies reported cardiac troponin levels within 48 hours of entering the ICU. In these studies, cardiac troponin I was investigated. The heterogeneity among these studies was not significant . Based on meta-analysis, cardiac troponin levels were 0.23 ng/mL more in patients treated with anesthetized isoflurane than those given sevoflurane with anesthesia. This difference was not statistically significant . A forest plot is available in Four studies reported cardiac troponin levels within 24 hours of entering the ICU. Two of studies had reported the cardiac troponin I and two studies had reported cardiac troponin T. The heterogeneity among these studies was not significant  Based on meta-analysis, changes in troponin levels were 0.67 ng/mL more than in patients treated with an isoflurane-based drug than those treated with anesthetized sevoflurane. This difference was not statistically significant. )MD = 0.67; 95% CI = 0.43 to 0.90; P < 0.001). A forest plot is available in Three studies reported cardiac troponin levels within 48 hours of entering the ICU. Two of studies have reported the cardiac troponin I and one of the cardiac troponin T. The homogeneity between the studies was not significant . It also changes after 24 hours, the isoflurane group was 2.28 times higher than the sevoflurane group . In Sensitivity analysis was performed by removing one of studies.df = 2; P = 0.59).The base result of Egger regression test publication bias between studies in this meta-analysis was not significant  are of biomarkers for identification of myocardial injuries in patients undergoing cardiac surgery. High level of troponin is an index for this diagnosis.31 Volatile anesthetic, sevoflurane, and isoflurane in patients undergoing heart surgery creates hemodynamic stability and maintain anesthesia depth. Additionally, the properties of these drugs in preventing perfusion and the protective effect on myocardium have led to their use in heart surgery.32 Inhalational anesthetics such as sevoflurane have neuroprotective effects and protective effects on hepatocytes as well.35With the increased prevalence of cardiovascular disease, millions of people are undergoing cardiac surgery.36Myocardial preconditioning during anesthesia is a cellular protective method in which exposure to a volatile anesthetic reduces cardiomyocyte injury. Preconditioning by sevoflurane has displayed cardiac protective effects on hypoxia/reoxygenation injury, but the related mechanism is uncertain. In a study syntax in 1A (STX1A), an essential regulator in cardiac disease was considered to be the target gene of microRNA-34a-5p.37The myocardial protective effect of sevoflurane may be related to the enhancement of mitochondrial respiratory function of cardiomyocyte after upregulation of HIF-1α gene expression.This meta-analysis was performed with 5 studies in 381 patients undergoing cardiac surgery for the evaluation of cardiac troponin during the use of anesthetics of isoflurane and sevoflurane. The results of this study showed changes in levels of troponin in patients entering the ICU. The level of troponin in patients who were under anesthesia with isoflurane increased less than the level of troponin in patients who were with anesthetics sevoflurane. Based on the subgroup analysis results, level of troponin T and I in the patient under cardiac surgery with isoflurane was less. The mean difference in troponin level between the two groups of isoflurane and sevoflurane was not significant 6 hours, 12 hours, and 48 hours after entering the ICU. However, sevoflurane had a better effect on troponin level 24 hours after surgery. However, according to the results of subgroup analysis, sevoflurane and isoflurane, respectively, had better effects on troponin T and I.In our study, the mean difference of troponin levels was also calculated using sevoflurane and isoflurane 24 and 48 hours after surgery, as compared to the time in ICU. Within 24 hours after surgery, the troponin level of sevoflurane was lower. However, there was no significant difference between the two drugs. Also, for troponin T and I, the level of sevoflurane was lower than isoflurane. In subgroup analysis, there was no difference in the level of troponin T and I between the two drugs. 48 hours after the surgery, the increased troponin levels were lower with sevoflurane compared with isoflurane. In subgroup analysis, based on the type of troponin, sevoflurane had a better effect on the type of troponin I and isoflurane had a better effect on the type of troponin T.40 Studies have also been conducted on troponin levels in patients undergoing cardiac surgery. Moreover, in all of these studies, there were contradictions similar to our study. Some reported that there was no difference between the two drugs, while others reported superiority of one over the other. For example in the study conducted by Parker et al19 and Bennett et al,41 it was shown the protective effect in sevoflurane and isoflurane was similar and no changes were observed in troponin levels using the two drugs. Two studies42 reported that sevoflurane provides better hemodynamic and protective conditions in the heart.Overall, the results of the GRADE showed that our studies of strength had moderate evidence. Therefore, perhaps the cause of the difference between the results of troponin with isoflurane and sevoflurane at the times of this meta-analysis was the differences in the type of operation as well as the different hemodynamic conditions of patients undergoing cardiac surgery. In the past, some studies on inhaled anesthetic agents were performed to investigate their effects on myocardial protection.18 which was performed to compare the sevoflurane and isoflurane, sevoflurane had a better protective effect on the heart. Sevoflurane had a better effect on reducing the amount of troponin T in 24 hours after surgery. In the study conducted by Passaint et al,11 in 30 children aged 2 to 6 years, troponin I in the isoflurane group was higher than that of sevoflurane. Sensitivity analysis was 6 and 24 hours after ICU. When we removed Passaint et al11 study from the analysis, there was a small change in the amount of pooled relative to when we entered this research.In the study by Venkatesh et al39 In this meta-analysis, we encountered a few limitations such as the small number of clinical trials and low sample size. Moreover, some of the studies had not reported troponin levels in each group.As we found in the present study, differences and similarities in our research results are consistent with previous studies. However, in general, isoflurane and sevoflurane do not have a significant negative effect on troponin levels. Moreover, in the two previous meta analytic studies which had focused on the comparison of isoflurane and sevoflurane with propofol, it has been shown that inhalation anesthetics such as isoflurane and sevoflurane have an acceptable protective effect on myocardial infarction in comparison with propofol.This meta-analysis showed that blood troponin levels were significantly lower Arrival in ICU with isoflurane and 24 hours after ICU with sevoflurane in patients who underwent cardiac surgery. Generally, given the small mean difference between isoflurane and sevoflurane, it seems that isoflurane and sevoflurane do not have a negative effect on cardiac troponin level.None.The Ethics panel of the School of Health Management and Information Science Iran University of Medical Sciences indicated that ethics approval was not required for this systematic review.This study was funded by the School of Health Management and Information Science Iran University of Medical Sciences (code: 691). School of Health Management and Information Science did not involve in study design; in the collection, analysis and interpretation of data; in the writing of the report; and in the decision to submit the article for publication.Supplementary file 1 contains Search Strategy For Systematic Literature Review And Forest Plot Figures For Sensitivity Analysis.Click here for additional data file.


Determining whether associations between gut microbiota characteristics and host physiology represent causal relationships is a fundamental challenge for microbiome research. We report a detailed investigation of microbiome assembly in C57BL/6 germ-free mice across a period of 70 days and compare the effects of single and multiple rounds of gavage, using both native and antibiotic-disrupted murine donor material. Recipients of the native microbiota did not achieve compositional stability until day 28 and persistent differences to donor microbiota remained until day 70. Performing multiple rounds of gavage significantly increased the cumulative number of detected taxa (mean increase: 10.4%) and compositional similarity to donor, and significantly reduced within-group variance (p < 0.05). Multiple rounds of gavage with antibiotic-disrupted microbiota provided no substantial benefit in relation to compositional similarity to donor or within-group variance. The process of donor microbiota establishment in recipient animals is necessary before experimentation commences and is considerably influenced by donor microbiota characteristics.  •Gut microbiota in germ-free mice stabilize 28 days after native microbiota gavage•Repeated gavage of native microbiota increases similarity to donor microbiota•Establishment of antibiotic-disrupted microbiota does not improve with repeated gavage•Donor microbiota characteristics influence fidelity of microbiota re-establishment Biological Sciences; Microbiology; Microbiome Much of our understanding of the influence of the gut microbiome on human physiology is derived from observational studies. However, where associations between microbiome traits and host phenotype are identified, it then becomes necessary to determine whether these relationships are causal, secondary to observed physiological phenomena, or result from parallel but independent processes.One of the few experimental strategies available to investigate causality in host-microbiome interactions is to instil intestinal microbiota into germ-free mice and assess whether donor phenotype is recapitulated. Such a strategy is becoming increasingly popular due both to growing access to gnotobiotic facilities and to evidence that antibiotic depletion of intestinal microbiota in conventional mice prior to fecal instillation represents a relatively poor alternative . AdditioDespite the utility of gut microbiota transplantation into germ-free mice as a means to understand microbiome-host relationships, there is little consistency in the manner in which the technique is performed, either for the instillation of intestinal microbiota from animal models  or humanMany of the bacterial clades that are closely associated with the regulation of host physiology are obligate anaerobes  and are A number of previous studies have aimed to describe the process of intestinal microbiota assembly in germ-free mice . In partWe investigated the dynamics of donor microbiome assembly in the gut of recipient germ-free mice, including a comparison of the effects of single and multiple rounds of gavage . In addiLactobacillus, Turicibacter, Faecalibaculum, Bifidobacterium, and Enterorhabdus were predominant in the donor native microbiota but were depleted in the antibiotic-disrupted microbiota , but higher evenness  and diversity  D. Bactercrobiota E. In con6 (standard deviation (SD): ± 4.3 x106) bacterial cells/mg feces. In mice that received antibiotic-disrupted microbiota, the pattern of an increase in bacterial load following instillation was similar to that seen with native microbiota, with the D14 to D70 average fecal bacterial load at 6.6 x 106 (SD: ± 3.1 x106) bacterial cells/mg feces  peaked between day four (D4) and day seven (D7) after the initial gavage, before declining to D14 A. From Dmg feces B. The toThe evenness and diversity (Faith's phylogenetic diversity) of the microbial community in recipients that received single or multiple gavages of the native microbiota, were higher when compared to the donor inoculum at all timepoints A and S3BInitial assessment of donor-recipient microbiota similarity was based on the percentage of donor taxa that were detectable in recipient feces. Similar trends were observed for recipients of one and three rounds of gavage, with detected taxa rising steeply from D4 : 1G = 43.1%, 32.8–44.0; and 3G = 44.8%, 40.5–44.8) to D14  A. At D21The percentage of donor taxa detectable in recipient animals fluctuated over the 70-day period of assessment A. As micRecipient-donor microbiota similarity was assessed based on weighted UniFrac distance . BroadlyLactobacillus and uncultured members of Muribaculaceae, respectively.Temporal changes in taxon relative abundance differed substantially between phylogenetic clades. For native microbiota, higher donor-recipient compositional distances were observed between D4 to D21 compared to later timepoints, while compositional variation in the single gavage recipients was also higher compared to those receiving multiple gavages. During this period of instability, variations between the single and multiple gavage groups were driven by alterations in the relative abundance of Bacteroidales, Lactobacillales, Erysipelotrichaceae, Verrucomicrobiales, Lachnospiraceae, Bifidobacteriaceae, Coriobacteriales, and Peptostreptococcaceae (p < 0.05) . The relLess variation in bacterial family relative abundance were observed in recipients of antibiotic-disrupted microbiota . The relBlautia (Lachnospiraceae), Bifidobacterium (Bifidobacteriaceae), and Akkermansia , which were the dominant members of bacterial families that showed substantial relative abundance changes in recipients of native or antibiotic-disrupted microbiota  was used to determine the absolute abundance of three genera, crobiota . The dynBifidobacterium, Clostridaceae, and Peptostreptococcaceae, can strongly influence fecal pH, which in turn can influence the ability of other gut bacteria to grow  to produce lactate, which reduces fecal pH (Escherichia (phylum Proteobacteria) as reported previously , Lachnospiraceae UC5-1-2E3, and Mollicutes RF39. Taxa within the Bacillales, Peptococcaceae, and Mollicutes RF39 order also peaked in relative abundance by D14 to D35 onward in recipients of multiple gavage with the native microbiota but not in single gavage recipients. Multiple gavages was also associated with reduced inter-animal variance, an important factor when using such animals to investigate host-microbiota interactions.Approaches used in the colonization of germ-free mice as part of published studies vary considerably. Some investigators have employed single rounds of gavage , while oAnaerofustis, Bacteroides, and Erysipelothrix. Why such differences should occur between mice of similar genetic backgrounds is not clear; however, there are a number of potential contributory factors. For example, the donor transplant material were derived from the cecum, while recipient microbiota composition was assessed based on fecal pellets. Differences in gut physiology in gnotobiotic mice compared with conventional mice have also been described, including in immune mechanisms that are involved in the regulation of intestinal microbiology , or present at low abundance (<1%) . Compared to native microbiota, instillation of antibiotic-disrupted microbiota was associated with greatly reduced temporal variation. Relative abundance of the predominant taxa, including Lachnospiraceae, Bacteroidales, and Verrucomicrobiales, achieved levels comparable to those in donor material upon instillation and showed little variation over time. Divergence from donor composition was driven by a failure to recover less prevalent taxa, including Staphylococcus, Clostridiales vadin BB60, and Alistipes , as well as multiple members of the Lachnospiraceae  and Ruminococcaceae families (Rumimiclostridium) also failed to colonize recipient animals by D4 (either single or multiple gavage). The genera Intestinimonas and Anaeroplasma, which are of the Ruminococcaceae and Anaeroplasmatales families, respectively, were detected only in multiple gavage recipients. As these taxa were still detected in the fecal microbiota of recipients that received the native microbiota, it is unlikely that the failure to detect these taxa in the antibiotic-disrupted recipients were due to selective characteristics of the cecum and luminal colon in the donor and recipient mice, respectively. The detection of these taxa in the donor could represent bacterial taxa that were largely nonviable due to antibiotic exposure. Failure of selected taxa to re-establish in the recipient can also be attributed to competitive interactions and compensatory relationships between bacterial species. The re-establishment of selected donor taxa can be inhibited by novel interactions that form within an altered microbial community to generate an alternate stable equilibrium , subject to similar environmental and dietary exposures. There is a growing number of studies involving the instillation of human stool, or stool-derived microbiota, into gnotobiotic mice. Substantial differences in genetics , age Le, type ofIn summary, a substantial period (more than 4 weeks) may be required following fecal instillation into germ-free mice to achieve closest compositional similarity to the donor for the native microbiota. Establishment of donor microbiota occurs more rapidly in less diverse bacterial communities that result from antibiotic exposure. The process of microbiota assembly differs considerably based on the complexity and composition of donor bacterial communities. Multiple rounds of fecal instillation result in greater similarity to donor microbiota for the native microbiota. Using the same regimen for microbiota instillation involving an altered microbial community did not improve donor-recipient microbiota similarity. A failure to understand the extent to which donor microbiota has been established in recipient mice and the degree of community stability achieved could contribute to spurious findings.Our study had a number of limitations. The dynamics of microbiota changes at the species level could not be assessed, as the highest resolution of taxa classification by 16S rRNA sequencing is to the genera level. We performed donor taxa representation by longitudinal fecal microbiota assessments of the recipients against the donor cecal material, which may influence taxa detection due to site differences. We also did not assess all possible gavage schedules, donor/recipient microbiota variants, cage arrangements, age and gender specificities but rather examined overall features of microbiota establishment in recipients. Microbiota assembly dynamics are likely to change with donor microbiome characteristics and can therefore vary with mouse genetic background  and betwjocelyn.choo@sahmri.com)Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Jocelyn Choo (The study did not generate new unique reagents.The accession number for the sequence data reported in this paper is SRA: PRJNA592263.All methods can be found in the accompanying


The aseptic meningitis caused by varicella zoster virus (VZV) reactivation was less described in the literature, most of which were detected by means of polymerase chain reaction. The authors presented 4 adult immunocompetent patients with acute aseptic meningitis with VZV infection diagnosed by next-generation sequencing (NGS).2O; range 165–400 mm H2O). White blood cell counts and protein levels were significantly elevated in CSF samples . The cytology of CSF demonstrated a lymphocytic pleocytosis, and most multinuclear cells. The culture of CSF was negative for all 4 cases, while T-cell spot test was positive for 2 cases, who were administrated with anti-tuberculosis treatment for suspicious tuberculous meningitis. NGS of CSF  detected specific sequences of VZV in the 4 cases within 72 h after admission. The inappropriate treatment were stopped while acyclovir were continued intravenously for 10–14 days. All patients recovered completely.Four patients were admitted to the hospital with headache and fever between March 2018 and August 2019. The median ages were 37 years (range 22–52 years). The median symptoms onset to clinic time was 3.5 days (range 3–6 days). Two patients had signs of meningeal irritation. Rash occurred after the meningitis symptoms in 1 patient . No other sign or symptom was reported. The brain Magnetic resonance imaging and electroencephalography were normal in all patients. Cerebrospinal fluid (CSF) samples were obtained at a median of 4 days (range 3–7 days) from the meningitis symptoms onset. Opening pressure of lumbar puncture after admission were high in these cases (median 256 mm HVZV is an infectious agent that causes aseptic meningitis in immunocompetent adults and could not be accompanied by skin manifestations. The NGS of CSF is a rapid detection for the identification and differentiation of meningitis in patients, which is of great importance for providing the rapid and accurate diagnosis and the targeted antimicrobial therapy for central nervous system infection. Aseptic meningitis is an inflammation of the meninges associated with acute onset of headache, fever and neck stiffness, with pleocytosis of the cerebrospinal fluid, and no growth on routine bacterial culture . The leaWe here described cases of 4 immunocompetent adults with aseptic meningitis due to VZV reactivation diagnosed by next-generation sequencing (NGS).On March 2018, a 52-year-old man was admitted to Northern Jiangsu People’s Hospital because of headache for 4 days and fever (37.6 °C) for 2 days. He had no other symptoms. The constant and progressive headache did not relieved after the routine treatment such as oral nonsteroidal analgesics. He had a history of chicken pox when he was 7 years old. He had symptomatic epilepsy since 8 years ago after a brain trauma, which occurred every 6 to 12 months. He had no sign of meningeal irritation, and vital signs were normal. No rash was seen at any stage  contained 147 × 10^6/L white blood cells (WBCs) ; CSF protein was 1.72 g/L   were continued intravenously for 10 days. The patient’s condition improved quickly. A repeat lumbar puncture on the ninth day after admission revealed improved WBCs (39 × 10^6/L) and protein (0.88 g/L) Table . Thus heOn November 2018, a 22-year-old man was admitted to Northern Jiangsu People’s Hospital because of fever (37.7 °C), severe headache, and nausea for 3 days. One day before admission, he noticed vescicles in a very limited region of the left dorsal skin. No other sign or symptom was reported . The cytology of CSF demonstrated WBCs with 200 × 10^6/L, and an increased protein concentration (0.57 g/L)  was continued intravenously for 10 days. The therapy was highly effective and the patient’s clinical condition rapidly improved. The CSF on the eighth day after the admission revealed WBCs with 130 × 10^6/L, and protein (0.40 g/L) Table . The pat2O). The CSF contained 478 × 10^6/L WBCs; CSF protein was 1.79 g/L  and protein (0.65 g/L) Table . Thus he2O, and the CSF contained 434 × 10^6/L WBCs , and normal protein (0.37 g/L)  and a progressive headache was admitted to Northern Jiangsu People’s Hospital. His disease began with headache and nausea 3 days earlier. No other symptom was developed. Neurological examination was moderate for neck stiffness and negative for Kernig signs  and VZV-R2 (GCGGTAGTAACAGAGAATTTCTT). The results showed that the read from Sanger sequencing was consistent with VZV genome  and herpes zoster. Varicella usually results in mild to moderate illness in mainly childhood or immunocompetent patients with disseminated vesicular rash. After primary infection, VZV remains latent in sensory cranial nerve ganglia or dorsal root ganglia, when reactivated, replicates along the course of the nerve and appears as a localized vesicular skin rash. Viral reactivation can cause a wide range of neurologic disease, most frequently manifesting as herpes zoster and post-herpetic neuralgia . Older aThe etiology of acute meningoencephalitis remains undiagnosed in approximately 60% of cases despite extensive clinical laboratory testing for infectious pathogens . Till no2O) in three patients, and it was also on the high side (165 mm H2O) for the other one. All cases showed elevation in CSF WBC, ranging from 147 to 478 × 106/L, and the CSF cytology indicated lymphocytic inflammation. It was regarded that mild elevation in CSF protein levels would been observed in aseptic meningitis. However, the relatively high CSF protein levels were reported seen in patients with VZV infection which were significantly higher than those seen in patients with enteroviral infection [Lumbar puncture revealed increased opening pressure of CSF (235–400 mm Hnfection , 13. ProThe difficulty in meningitis diagnosis is to distinguish whether it’s a viral or bacterial etiology, because this is crucial for treatment decisions. The treatment threshold is usually set low in clinical work, so that antibiotics, even anti-tuberculosis treatment, are often prescribed in cases of doubt, as were patients in this study. The patient of case No. 2 had vesicular rash before admission which made the diagnosis with aseptic meningitis relatively easy. However, the other 3 patients never showed cutaneous zosteriform lesions. Additionally, the T-cell spot test was positive for the second and fourth cases, meanwhile the protein levels in CSF elevated significantly for both these 2 patients, which implied possible tuberculosis infection, therefore anti-tuberculosis treatment as well as antibiotics were administrated. However, all the cases in our study were pathogen diagnosis within 72 h after admission. Once the sequences of VZV were detected by NGS, inappropriate treatment were stopped.NGS is a rapid and accurate approach for the molecular diagnosis with diseases compared to traditional clinical testing. It could dramatically reduce the diagnostic period to less than 3 days . PathogeVZV reactivation leading to aseptic meningitis in immunocompetent adults with or without cutaneous zoster is more common that previous regarded. Relatively high CSF protein levels could be observed in VZV meningitis. This study highlighted the feasibility of using NGS of CSF as a diagnostic tool for CNS infection. Unbiased NGS could facilitate identification of all the potential pathogens in a single assay theoretically, which is of great importance for providing the rapid and accurate diagnosis and the targeted antimicrobial therapy for CNS infection.Additional file 1. Details of NGS detection process and the results of Sanger sequencingAdditional file 2. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 1Additional file 3. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 2Additional file 4. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 3Additional file 5. Microbe reads of bacterium, fungi, parasite and virus detected in Case No. 4Additional file 6. Original images for results of Sanger sequencing detection of VZV from CSF specimen


Among all CXCL13 groups, their results showed no correlation between CSF CXCL13 concentrations and serum CXCL13 or Qalbumin. The authors argue that, in contrast to other proteins, CXCL13 passage across the BCSFB does not occur, regardless of BCSFB function, and is instead solely influenced by intrathecal production. In contrast to the authors’ findings, in our studies including both non-inflammatory neurological disorders  and multiple sclerosis (MS) patients we observed a significant correlation between serum CXCL13 concentrations and CSF CXCL13 concentrations. We review several observations which may underlie these contrasting results, including (1) the impact of serum CXCL13 concentrations on CSF CXCL13 in patients with lower intrathecal CXCL13 production and thus lower CXCL13 concentrations (i.e. NIND and MS), (2) the proposed diffusion dynamics of the small molecule CXCL13 across the BCSFB, and (3) differing definitions of negative versus elevated CSF CXCL13 concentrations determined by an assay’s relative sensitivity. In conclusion, we argue that for patients with moderately elevated CSF CXCL13 concentrations, serum CXCL13 concentrations influence CSF CXCL13 levels, and thus the appropriate corrections including incorporation of CSF/serum ratios and Qalbumin values should be utilized.Pilz et al.  investigated how CSF CXCL13 concentrations are influenced by CXCL13 serum concentrations and blood-CSF barrier (BCSFB) function, comparing the impact of serum CXCL13 levels and Q There has been increased attention to candidate biomarkers in the cerebrospinal fluid (CSF) in neurological diseases. For biomarkers derived from the periphery or CNS, including inflammatory proteins, the relationship between serum and CSF levels of these proteins are an important concept. CXCL13, a 10 kD chemokine, is a candidate CSF biomarker in MS , 3. CXCLPilz et al.’s conclusion that the BCSFB is completely closed to a small protein like CXCL13 runs counter to long experience of neuroscientists with the equilibrium between proteins in the blood and CSF , and wouCXCL13, i.e. the ratio of CSF to serum CXCL13, in “normals” and non-inflammatory controls, is approximately 0.025–0.1 [Second, the conclusions in the Pilz manuscript were not sufficiently supported by the data presented, especially for inflammatory conditions, such as multiple sclerosis (MS), where there is minimal, if any, BCSFB dysfunction, and there are small elevations of intrathecally produced CSF CXCL13 relative to the dramatic elevations seen in diseases such as Lyme neuroborreliosis (LNB) –12. ThusIn MS, there is intrathecal production of CXCL13, but at a lower level. Thus, the diffusion of serum CXCL13 into the CSF contributes a larger fraction of CSF CXCL13 than in LNB or the other highly inflammatory conditions, such as meningitis and encephalitis, in the Pilz study. Thus, utilizing the CXCL13 index, which accounts for both serum concentrations and BCSFB function, provides better information than CSF CXCL13 concentrations alone [CXCL13 for CXCL13 would be approximately 0.025–0.1, and this number has been confirmed in a number of studies [Third, the prediction would be that, based on its molecular weight of 10.3 kDa, the Q studies , 15. TheFourth, Pilz et. al. were somewhat limited in analysis of situations with relatively low CSF CXCL13 by their use of the Euroimmun CXCL13 ELISA as their method of measurement. This ELISA, which is widely used for LNB , 17, is Finally, there is strong evidence from our laboratory in non-inflammatory neurological conditions (NIND) that blood levels of CXCL13 contribute to CSF concentrations. Unlike the chemokines CXCL10, CCL2, CCL19, and CXCL12, among others, which are constitutively produced in the CNS , 19–21, In conditions in which intrathecal production of CXCL13 is exuberant, such as in LNB and other highly neuroinflammatory conditions, the contribution of serum CXCL13 to CSF CXCL13 concentrations is relatively slight, and is dwarfed by intrathecal production, but in many other conditions such as in MS and in non-inflammatory neurological diseases , diffusi


Allogeneic hematopoietic cell transplant (HCT) is curative for pediatric patients with non-malignant hematopoietic disorders, including hemoglobinopathies, bone marrow failure syndromes, and primary immunodeficiencies. Early establishment of donor-derived innate and adaptive immunity following HCT is associated with improved overall survival, lower risk of infections and decreased incidence of graft failure. Immune reconstitution (IR) is impacted by numerous clinical variables including primary disease, donor characteristics, conditioning regimen, and graft versus host disease (GVHD). Recent advancements in HCT have been directed at reducing toxicity of conditioning therapy, expanding donor availability through use of alternative donor sources, and addressing morbidity from GVHD with novel graft manipulation. These novel transplant approaches impact the kinetics of immune recovery, which influence post-transplant outcomes. Here we review immune reconstitution in pediatric patients undergoing HCT for non-malignant disorders. We explore the transplant-associated factors that influence immunologic recovery and the disease-specific associations between IR and transplant outcomes. Allogeneic hematopoietic cell transplant (HCT) is a key therapeutic approach for many non-malignant hematopoietic diseases in pediatric patients, including hemoglobinopathies, bone marrow failure syndromes, and immunodeficiencies. Effective reconstitution of donor-derived innate and adaptive immune cell number and function following HCT is critical for promoting donor cell engraftment, restoring protection against infections, and improving overall survival , 2.Recovery of immunity after HCT is influenced by various clinical factors, including primary diagnosis, donor type, stem cell source, graft manipulation, conditioning regimen , and pharmacologic prophylaxis, development and treatment of graft-versus-host disease (GVHD) , 2. AftePeripheral blood (PB), bone marrow (BM), or umbilical cord blood (UCB) stem cells can be utilized for HCT from either related (RD) or unrelated donors (URD). These donor sources vary in cellular composition with PB grafts having 10-fold higher T and B cells than BM grafts and single UCB grafts having 10–100-fold fewer nucleated cells compared to BM , 5. The Graft source also impacts reconstitution of adaptive immunity. HCT with UCB has been associated with higher naive and memory B cell numbers at 6 months post HCT compared to BM and PB grafts . In contT cell reconstitution also differs between BM and PB recipients. In a single institution randomized trial, patients who received PB grafts had faster lymphocyte recovery, most significantly CD4 T cells, compared to BM graft recipients . Thus, dWhile an HLA matched donor is preferred, less than 25% of patients will have an available sibling donor and the likelihood of identifying a matched URD in the registry is impacted by numerous factors, including ethnicity of the patient . Consequex vivo expansion of cord blood units. While IR data on double UCB HCT is limited in pediatrics, in adults, it has not consistently demonstrated an improvement in IR compared to single UCB HCT  and reduced intensity conditioning (RIC) regimens in order to limit the morbidity associated with myeloablative conditioning (MAC). RIC regimens are non-myeloablative while RTC regimens are myeloablative. Both approaches have fewer side effects and organ toxicities compared to traditional MAC. Law et al. reported that following a RTC regimen of alemtuzumab, busulfan, and fludarabine median time to neutrophil recovery was 16 days while time to B cell and T cell reconstitution was 3 and 6 months, respectively . A RIC aTiming and dose of serotherapy significantly impact IR , 29, 30.+) recovery was delayed more than a year and correlated with age, with younger patients having faster recovery  HCT with BM graft . They forecovery . Interesrecovery .in vivo T cell depletion is increasingly utilized in patients with hemoglobinopathies  with aplastic anemia treated with haploidentical HCT utilizing busulfan, cyclophosphamide and ATG reported rapid neutrophil recovery at median of 12 days and monocyte recovery by 30 days after transplantation . CD8 T cIL2RG), Janus-associated kinase 3 (JAK3), and adenosine deaminase (ADA) mutations (IL2RG- and JAK3-mutant SCID transplanted without conditioning have lower (often absent) donor stem cell engraftment and, consequently, do not have donor B cell repopulation (IL7R)-deficient SCID have intact function of B cells, which can produce immunoglobulin with help from donor T cells  have arrest of thymopoiesis at later developmental stages and require conditioning to achieve recovery of donor immunity (Severe combined immunodeficiencies (SCID) are a heterogeneous group of genetic disorders characterized by a lack of T cell progenitors available to develop within the thymus resulting in failure of T cell maturation as well as impaired cellular and humoral immunity . IR follutations . Howeverpulation . In the  T cells . Notably T cells . In ADA-moral IR . SCID paimmunity .A recent prospective study demonstrated that patients with SCID who received conditioning (RIC or MAC) prior to HCT had significantly higher levels of T, B, and myeloid cell donor chimerism at day 100, which persisted at 1 year post HCT . FurtherRAG1/2 and DCLRE1C mutations have poorer T cell reconstitution after transplant , keratinocyte-growth factor, sex steroid ablation, and adoptive cell therapies , 46–51. SB and JB wrote the manuscript jointly. Both authors contributed to the article and approved the submitted version.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Testicular prostheses: development and modern usage. Ann R Coll Surg Engl. 2007 May; 89(4):349-53. doi:J Pediatr Urol. 2016; 12(6):335-343. doi:10.1016/j.jpurol.2016.07.014Radmayr C, Dogan HS, Hoebeke P, et al. Management of undescended testes: European Association of Urology/European Society for Paediatric Urology Guidelines . Three references were added –4:XimenaThe article titled “Impalpable Testis: Evaluation of Diagnostic and Treatment Procedures and Our Treatment Protocol”  was founAbstractIntroduction. Cryptorchidism is one of the most frequent congenital malformations in male newborn. The aim of this paper is to present current literature recommendations and our treatment protocol of impalpable testis based on our experience. Material and methods. This is a retrospective study where we analyzed clinical documents from January 2010 to December 2015. We reviewed diagnostic procedures, intraoperative findings with closing diagnosis, and all treatment modalities for patients with impalpable testis. We also investigated current literature regarding this topic. Results. Ninety-one patients were admitted under the diagnosis of impalpable testis. In 39 patients, ultrasound detected testis in the inguinal canal and standard open orchidopexy was done. In 25 patients, laparoscopy showed (48.08%) the entrance of the spermatic cord into the inguinal canal, then open exploration of canal was performed, testicular remnant removed, and simultaneously appropriate testicular prosthesis was implanted. Next, twenty patients underwent orchiopexy of the abdominal testis. Four of them underwent the Fowler-Stevens procedure in two stages, and in 16 patients, deliberation of the testis was sufficient to place the testis into the scrotum during the same operation. Conclusions. Improvements in therapeutical protocol, with additional clinical examination in anesthesia, switch to initial inguinal approach and laparoscopic exploration of the abdomen in case of negative finding in the inguinal canal could improve outcome for these patients. If testicular nubbin is found during exploration, its excision is highly recommendable, as well as implantation of the testicular prosthesis at the same time during the surgical orchiectomy.Among all congenital malformations in boys at the time of birth, cryptorchidism is the most common . Although the exact etiology of this condition is unknown, it is believed to be the result of multiple factors . It affects preterm neonates much more than full-term boys (1.1 -45.0% vs. 1.0-4.6%) . The definitive diagnosis of undescended testis is made after six months of age due to the postnatal spontaneous testicular descent. However, nearly 1% of 1-year-old boys have the diagnosis of cryptorchidism . The most useful clinical classification is into palpable and nonpalpable testes. Almost 80% of undescended testes are palpable [4]. When considering anatomical location, nonpalpable testis generally could be absent due to prenatal regression (vanishing testis), agenesis (true monorchia), and located in the abdomen or in inguinal canal. Furthermore, testis could be located anywhere outside its normal path of descent when it is called ectopic testis . It is crucial for appropriate therapy to make precise diagnosis at a timely manner (6 to 12 months) . Once the diagnosis of impalpable testis has been made, a suitable treatment protocol should be strictly followed. An optimal treatment protocol is however a matter of debate in the current literature. In this paper, we would like to present our latest treatment protocol.From January 2010 to December 2015, 493 patients with undescended testis underwent surgery at the Institute for Child and Youth Healthcare of Vojvodina. Ninety-one patients (18.5%) had impalpable testes. A retrospective review was conducted. Data that we collected were diagnostic procedures used for a confirmation of a diagnosis of impalpable testis. Intraoperative findings, final diagnosis, treatment modality, and outcome were noted for every patient. This study has the approval of the Institutional Ethical Board.Patients with impalpable testis and negative ultrasound underwent abdominal laparoscopy. Open Hasson technique with infraumbilical incision was used to gain entry to the abdomen for laparoscopy, for insertion of the first port, and creation of pneumoperitoneum. If the testis is found intraabdominally, the two additional ports were inserted as well. They are so-called working ports. According to the finding, the surgeon could decide either to finish the laparoscopic procedure as one or two-stage Fowler-Stephens orchidopexy. In case with vas deferens and gonadal vessels entering the deep inguinal ring which means that testis or testicular remnant is present in the inguinal canal, standard inguinal exploration and eventually open orchidopexy were performed.In the case when no testis was identified or testicular remnant was present during inguinal exploration, the remnant was removed and an adequate testicular prosthesis was implanted simultaneously . The finP value that was 0.05 or less was used for the determination of statistical significance.We used the SPSS program for statistical analysis. Categorical data were compared using the Fisher exact test. While discussing results, Ninety-one patients were admitted under the diagnosis of impalpable testis. All patients underwent an ultrasound examination that revealed an inguinal testis in thirty-nine patients. All of those patients underwent inguinal orchiopexy.In 52 patients, ultrasound did not detect testis in the inguinal canal. Those patients were treated for impalpable testis by laparoscopy. Patients were 1-17 years old (average 3.83). Forty-three patients (82.69%) had unilateral impalpable testis, eighteen of these patients had right-sided (41.86%), and 25 had left-sided impalpable testis (58.14%). Nine patients (17.31%) had bilateral impalpable testes with either positive or unclear stimulation test. Twenty-five patients (48.08%) underwent laparoscopic exploration that confirmed the entrance of the spermatic cord into the inguinal canal, followed by open exploration, removal of testicular remnant and implantation of appropriate testicular prosthesis  underwent orchidopexy of the abdominal testis (46.51%), 4 of which underwent Fowler-Stevens procedure in two stages, and in 16 patients, deliberation of the testis and spermatic cord was sufficient to place the testis into the scrotum in one operation. One of the patients with bilateral abdominal testes was operated in a single-stage laparoscopy procedure bilaterally, but required a subsequent open reorchidopexy on one side. In 7 patients (7/52), laparoscopy confirmed the entrance of the spermatic cord into the inguinal canal, where in three patients we found hypoplastic testis that we decided to leave in place, and in the other 4 patients, the remnant was removed, but prosthesis was not implanted either due to a lack of the appropriate size or parental consent. Therefore, in these seven patients we did not place the prosthesis during the same operation.Our treatment protocol and results are presented in At the Institute for Child and Youth Healthcare of Vojvodina, a child with undescended testis is followed from birth. During that time, the clinical exam is performed minimally three times: days after birth, at 6 and 9 months of age. The finding of enlarged contralateral testis could suggest absent or atrophic testis ipsilaterally . However, that should not withdraw the patient from surgical exploration of the abdomen and/or inguinal canal . If testis is clinically impalpable, an ultrasound examination is always performed. Ultrasound can confirm the presence of almost 80% of all testes located in the inguinal canal with an accuracy of more than 90% . The high precision suggests that inguinal exploration and orchidopexy could be performed without diagnostic laparoscopy . In any case, diagnostic and therapeutic laparoscopy has become a modern useful asset or a new “gold standard” for treating patients with impalpable testis .Surgeons should always use a chance to perform one more clinical examination during general anesthesia [2]. Sometimes, the previously impalpable testis is found and the planned laparoscopic operation could be cancelled. Then, orchidopexy is performed using a classical open approach . Literature suggests that a wide range of around 20% to 85% of inguinal testis could be found during surgery in patients previously diagnosed with nonpalpable testis . This kind of examination under general anesthesia is not a standard part of our treatment protocol due to organizational issues. Namely, both a child and an operating theater are prepared for either conventional or laparoscopic surgery. It is however one of the things that we are planning to change in our protocol.The standard management of impalpable testes is primarily surgical. According to the newest literature, orchidopexy should be performed by the age of 6 months as it may improve the fertility potential and prevent possible tumor formation . Therefore, we tend to perform surgery early enough, but we also wait a little bit longer compared to the timing of treatment for patients with palpable undescended testis . The reason for this short delay is enabling the scrotal sac to grow enough to receive a testicular prosthesis.According to our treatment protocol, we start with laparoscopic examination which allows accurate diagnosis for intraabdominal testis [14], subsequent testicular nubbin removal, or one or two-stage Fowler-Stevens procedure. On contrary, there are some surgeons who prefer open inguinal exploration and after that planned laparoscopy for undiscovered testis . After years of practice, it might be advisable to consider the inguinal approach, together with examination under anesthesia as a first line of treatment. In that way, we would avoid laparoscopies that might be unnecessary and time-consuming, especially in obese children. Another controversial issue is the need for obligatory removal of testicular nubbin [16]. The treatment of patients with testicular regression syndrome (TRS) is controversial because 0 to 16% of them have viable germ cells [17-23]. If left in place, testicular remnant could become malignant according to one case reported in literature [24]. Although scientific conclusion should not be derived from one study alone, we also believe that testicular remnant should be excised as in 25% of cases seminiferous tubules are found during histological analysis and in 10% germ cells are found as well.Having an undescended testis is for many boys and even older men psychological issue [25-27] that could be partially solved by implantation of testicular prosthesis. As prosthesis does not grow with the child, it is of great importance to plan the correct timing for surgery [27]. If performed too early, it may lead to another surgery for a larger prosthesis. On the other hand, it prevents possible problems with undeveloped scrotum in adolescence which might not be suitable for larger testicular prosthesis insertion [27].We believe that recent improvements in our protocol, including introducing the additional clinical examination in anesthesia and switch to an initial inguinal approach, hold promise of improved outcomes. Even when inguinal exploration is being done, it is advisable to be ready to perform laparoscopic exploration of the abdomen in case of negative finding in the inguinal canal. Excision of the testicular nubbin is in our experience highly recommendable, as well as implantation of the testicular prosthesis at the time of orchiectomy.ReferencesActa Paediatrica, vol. 96, pp, 611–616, 2007, doi:10.1111/j.1651-2227.2007.00241.x.1. H. E. Virtanen, R. Bjerknes, D. Cortes, N. Jørgensen, E. Rajpert-de Meyts, A. V. Thorsson, J. Thorup, and K. M. Main, “Cryptorchidism: classification, prevalence and long-term consequences,” Journal of Pediatric Urology, vol. 12, no. 6, pp. 335–343, 2016. doi:10.1016/j.jpurol.2016.07.014.2. C. Radmayr, H. S. Dogan, P. Hoebeke, R. Kocvara, R. Nijman, S. Silay, R. Stein, S. Undre, and S. Tekgul, “Management of undescended testes: European Association of Urology/European Society for Paediatric Urology Guidelines,” International Journal of Andrology, vol. 31, pp. 1–11, 2008, doi:10.1111/j.1365-2605.2007.00770.x.3. K. Sijstermans, W. W. Hack, R. W. Meijer, and L. M. van der Voort-Doedens, “The frequency of undescended testis from birth to adulthood: a review,” Pediatric Endocrinology Reviews, vol. 11, pp. 240–250, 2014.4. C. Kollin and E. M. Ritzen, “Cryptorchidism: a clinical perspective,” The Journal of Urology, vol. 148, pp. 632–634, 1992, doi:10.1016/S0022-5347(17)36675-2.5. D. A. Diamond and A. A. 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It was found that in human umbilical vein endothelial cells (HUVECs) cultured with normal 5.5 mM or high 33 mM glucose, acacetin (0.3–3 μM) exerted strong cytoprotective effects by reversing high glucose-induced viability reduction and reducing apoptosis and excess production of intracellular reactive oxygen species (ROS) and malondialdehyde in a concentration-dependent manner. Acacetin countered high glucose-induced depolarization of mitochondrial membrane potential and reduction of ATP product and mitoBcl-2/mitoBax ratio. Silencing Sirt3 abolished the beneficial effects of acacetin. Further analysis revealed that these effects of acacetin rely on Sirt1 activation by increasing NAD+ followed by increasing Sirt3, pAMPK and PGC-1α. In STZ-diabetic mice, acacetin significantly upregulated the decreased signaling molecules  in aorta tissue and attenuated atherosclerosis. These results indicate that vascular endothelial protection of acacetin by activating Sirt1/Sirt3/AMPK signals is likely involved in alleviating diabetes-accelerated atherosclerosis by preserving mitochondrial function, which suggests that acacetin may be a drug candidate for treating cardiovascular disorder in patients with diabetes.The strategy of decreasing atherosclerotic cardiovascular disorder is imperative for reducing premature death and improving quality of life in patients with diabetes mellitus. The aim of this study was to investigate whether the natural flavone acacetin could protect against endothelial injury induced by high glucose and attenuate diabetes-accelerated atherosclerosis in streptozotocin-(STZ) induced diabetic ApoE Since atherosclerotic cardiovascular disease is the leading cause of increased mortality and a major cause of morbidity in patients with Type 1 and Type 2 diabetes worldwide , decreasAlthough the details of the mechanisms responsible for the accelerated formation of atherosclerosis lesion observed in diabetes are not fully understood , it is g−/− mice induced by streptozotocin (STZ). Our results suggested that acacetin reduced vascular endothelial injury induced by high glucose and attenuated diabetes-accelerated atherosclerosis by Sirt1-mediated activation of Sirt3/AMPK signals.We have previously found that the natural flavone acacetin , in addition to its atrial-selective anti-atrial fibrillation property , is cardAcacetin and acacetin prodrug used in the present study were synthesized as described previously . Specifi2. For viability assay, cells were seeded in 96-well plates and grew to 70–80% confluence, then exposed to normal (5.5 mM) or high (33 mM) glucose (Sigma–Aldrich) medium for 5 days in the absence or presence of acacetin (0.3–3 μM). Cell viability was determined by 3--2,5-diphenyl-2-H-tetrazolium bromide (MTT)  assay as described previously  were obtained from ScienCell Research Laboratories  and cultured in plates pre-coated with 0.2% gelatin in endothelial cell medium (ECM) supplemented with 5% fetal bovine serum, 1% penicillin/streptomycin with 1% endothelial cell growth supplement (ScienCell) at 37 °C, 5% COeviously . Briefly+ production before high glucose exposure. The concentrations used were based on previous reports (In the experiments of HUVECs with inhibition of AMPK or nicotinamide phosphoribosyltransferase (NAMPT), the AMPK inhibitor dorsomorphin   was admi reports .Immunofluorescence analysis was used to identify Sirt3 expression levels in aortic root sections and cultured HUVECs with different treatment. Briefly, aortic root sections were stained with anti-Sirt3 and anti-CD31 antibody. Following an overnight incubation with primary antibodies, aortic root sections or cells were washed three times with phosphate buffered saline (PBS), followed by a 1-h incubation with Alexa Fluor–conjugated secondary antibodies  (Thermo Fisher Scientific) at room temperature, then mounted on DAPI-containing mounting media .HUVECs were plated on confocal culture dish and cultured, thereafter labeled with MitoTracker® Red CMXRos  incubated at 37 °C for 20 min, then washed with PBS three times and fixed in 4% paraformaldehyde  for 10 min. After permeabilization with 0.5% Triton X-100  for 10 min, the cells were blocked with 10% goat serum  at 37 °C for 1 h, then incubated with anti-Sirt3 antibody (Solarbio Technology) at 4 °C overnight. Images were captured with a laser scanning confocal microscope .Flow cytometry analysis was employed to assay the viability, apoptosis, ROS production and mitochondrial transmembrane potential in HUVECs using a flow cytometer  Flow cytometry analysis was employed to assay the viability, apoptosis, ROS production and mitochondrial transmembrane potential in HUVECs using a flow cytometer (Beckman Coulter). For viability and apoptosis determination, annexin V and propidium iodide staining were performed with the Annexin V-FITC apoptosis detection kit  following the manufacturer’s instruction, and the stained cells were subjected to flow cytometry within 1 h.For intracellular ROS analysis in HUVECs, 80-90% confluent cells seeded in six-well plate were pretreated with 0.3, 1, or 3 μM acacetin for 3 h, then exposed to 33 mM glucose medium with 0.3, 1, or 3 μM acacetin for 30 min, Afterward, the cell-permeable ROS probe 2′,7′-dichlorofluorescin diacetate  (Thermo Fisher Scientific) was applied and the cells were incubated for additional 30 min. After detachment with trypsin solution, the cells were washed with cool PBS for three times. Fluorescence intensity of DCFH-DA was determined in the single cell suspension using a flow cytometer (Beckman Coulter) with excitation wavelength of 488 nm and emission wavelength of 525 nm.For the determination of mitochondrial transmembrane potential, HUVECs exposed to 33 mM glucose 5 days in the absence and presence of 0.3, 1 or 3 μM acacetin were incubated with JC-1 (2 μM), a membrane permeable dye for probing mitochondrial membrane potential , at 37 °C for 30 min. Green fluorescence reflected the monomeric form of JC-1, and red fluorescence reflected the aggregate form. Mitochondrial membrane potential was then analyzed with a flow cytometer.HUVECs were cultured with 5.5 mM or 33 mM glucose (5 days) in the absence and presence of 0.3, 1, or 3 acacetin. The mitochondrial oxidative stress proteins (i.e. SOD activity and MDA content) of the harvested cells were measured using commercial Superoxide dismutase (SOD) Activity Colorimetric Assay Kit and kits and Lipid Peroxidation (MDA) Assay Kit  as described previously . The int+) and reduced nicotinamide adenine dinucleotide hydride  levels were quantified using an EnzyChrom™ NAD+/NADH assay kit  following the manufacturer’s instruction.HUVECs were vulture with 33 mM glucose for 5 days in the absence or presence of 3 μM acacetin or the NAMPT inhibitor GMX-1778 (10 nM). The harvested cells were rinsed with PBS twice and centrifuged 4 × at 1,000 rpm for 10 min, then oxidized nicotinamide adenine dinucleotide  using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific) following the manufacturer’s instruction. After 48 h transfection of control siRNA, Sirt1 siRNA or Sirt3 siRNA (Santa Cruz Biotechnology), the cells were incubated with 5.5 mM or 33 mM glucose culture medium in the absence or presence of 3 μM acacetin for 5 days, then collected for western blot analysis. The silencing efficiency of Sirt1 and Sirt3 proteins was significant, which is shown in Western blot analysis was employed to determine the expression of specific proteins in aortic tissues and cultured HUVECs. Proteins of aortic tissue homogenate lysates or HUVECs lysates prepared in sodium dodecyl sulfate (SDS) lysis buffer were extracted with RIPA buffer supplemented with protease and phosphatase inhibitors on ice, and protein concentration was determined using the BCA protein assay kit  as described previously . The pro−/− mice were obtained from Beijing Vital River Laboratory Animal Technology  and raised in Laboratory Animal Center of Xiamen University. The age-matched animals were cared for following the Guide for Care and Use of Laboratory Animals published by National Institutes of Health  of the United States. Type 1 diabetic mouse model was established in 7-week-old ApoE−/− mice by intraperitoneal injection of STZ  or vehicle citric acid (control) for five consecutive days. Random blood glucose was measured 2 weeks after last streptozotocin injection with the Accu-Chek Performa glucometer , and only animals with blood glucose >16.7 mM were classified as diabetic. Experiments were assigned as control, control with acacetin treatment, STZ-diabetes, STZ-diabetes with acacetin treatment. Animals with acacetin treatment received acacetin prodrug subcutaneously at 20 mg/kg twice daily (the prodrug can be metabolized into acacetin)  , and conAortic root lesion and en face lesion areas of whole aorta were fixed with 4% paraformaldehyde and stained with Oil Red O as described previously . Aortic p values <0.05 were considered statistically significant.Statistical analyses were performed with GraphPad Prism 6.0 . Results are presented as means ± SEM. One-way ANOVA followed by Bonferroni post hoc test were used for comparison among groups. To determine the potential protection of acacetin against vascular endothelial cells injury by high glucose conditions, HUVECs were cultured with normal glucose (5.5 mM) medium or a high glucose (33 mM) medium in the absence or presence of acacetin (0.3–3 μM). Acacetin had no effect on viability of HUVECs cultured with normal glucose medium , while iIt is generally believed that excess production of ROS is involved in endothelial apoptosis in diabetic cardiovascular complications. ROS production and the expression of antioxidant proteins SOD1 and SOD2 were therefore determined in HUVECs cultured with 33 mM glucose medium . High glp = 0.0038 vs. 5.5 mM glucose). Acacetin (3 μM) significantly countered the increase of mitoBax, the decrease of mitoBcl-2, mitoBcl-2/mitoBax ratio and mitoSirt3 induced by high glucose   and Bcl-2 (mitoBcl-2) in HUVECs . The HUVe alone) . Immunoce alone) . These rTo determine the potential role of Sirt3 in acacetin protection against high glucose-induced endothelial injury, siRNA molecules targeting Sirt3 were employed in HUVECs. Western blot analysis in HUVECs revealed that acacetin not only reversed the high glucose-induced decrease of Sirt3 expression in a concentration-dependent manner, but also the high glucose-induced downregulation of Sirt1, PGC-1α, and pAMPK expression . These rTo further identify the molecular target of acacetin for the protection against high glucose- or hyperglycemia-induced vascular endothelial injury, siRNA molecules targeting Sirt1 or Sirt3 and the AMPK inhibitor dorsomorphin were utilized in HUVECs cultured with 33 mM glucose to determine their effects on acacetin-induced upregulation of Sirt1, PGC-1α, Sirt3, and pAMPK ,B. It is+- dependent deacetylases that controls metabolism, and biosynthesis of NAD+ is mediated by NAMPT (+/NADH (+/NADH ratio and expression of Sirt1 and Sirt3 in the absence or presence of the NAMPT inhibitor GMX-1778 (CHS-828)  . The NADGMX-1778 . MoreoveGMX-1778  in HUVECpoptosis .−/− mouse model of STZ-diabetes. The lipid profiles of this diabetic model were illustrated in n = 8, p = 0.0021–0.00016 vs. control), while high-density lipoprotein was decreased by 33.1%  in STZ-diabetic ApoE−/− mice. Acacetin treatment did not alter the basal lipid profiles in control animals , and the percent decrease of high-density lipoprotein to 10.4% (p = 0.0043 vs. STZ-diabetes). However, the random blood glucose level was not significantly reduced in STZ-diabetic ApoE−/− mice treated with acacetin  and final bodyweight showed no differences between STZ-diabetic animals (25.91 ± 1.24 g) and STZ-diabetic animals treated with acacetin (27.87 ± 1.47 g)  . These r−/− mice than nondiabetic ApoE−/− mice (control), and the lesion was reduced in STZ-diabetic ApoE−/− mice treated with acacetin  in non-treated STZ-diabetic ApoE−/− mice to 7.0 ± 0.6% (p = 0.0071 vs. STZ-diabetes).The Oil Red O staining of en face aorta showed that atherosclerotic lesion was greater in STZ-diabetic ApoEacacetin . The anaacacetin  showed t−/− mice treated with acacetin . These results suggest that acacetin provides effective vascular protection against hyperglycemia-induced injury in STZ-diabetic ApoE−/− mice.The aortic root sections were stained with hematoxylin and eosin  or Oil R−/− mice, and STZ-diabetic ApoE−/− mice treated with acacetin . The alterations in the expression of these molecules were reversed in STZ-diabetic ApoE−/− mice treated with acacetin (p = 0.0399–0.0031 vs. STZ-diabetes). The immunostainings of aortic root sections, like in HUVECs cultured with high glucose, showed a decreased expression of Sirt3 in STZ-diabetic ApoE−/− mice, which were upregulated by acacetin treatment   , and is nd sKCa) . It alsond sKCa) , anti-pend sKCa) , and rednd sKCa) . The pre−/− mice and also in HUVECs cultured with high glucose medium.It is generally recognized that atherosclerosis is a multifactorial vascular progressive disorder involving alteration of several cellular and molecular events in diabetes . These i+ and NAMPT  located in nucleus , mitochondria , or cytoplasm (Sirt2) that are activated by NADnd NAMPT . Sirt1 ind NAMPT . The pre−/− mice.However, silencing Sirt1 significantly decreased expression of Sirt3, pAMPK and PGC-1α and completely abolishes acacetin-induced upregulation of these molecules in HUVECs cultured with high glucose medium, whereas the AMPK inhibitor dorsomorphin reduced expressions of Sirt3, pAMPK and PGC-1α and fully inhibited acacetin-induced upregulation of pAMPK and PGC-1α . AMPK inhibition does not affect Sirt1 expression or acacetin-induced upregulation. These results indicate that protection of acacetin against vascular hyperglycemic injury is related to Sirt1-mediated activation of Sirt3 and AMPK, followed by AMPK-dependent PGC-1α activation which regulates biosynthesis  and ATP +/NADH ratio. Furthermore, the water-solubility of acacetin is low (0.023 μM) and therefore oral bioavailability is poor. The issues of water-solubility and bioavailability of acacetin have been solved by synthesis of water-soluble prodrug for injection administration, which can be used clinically in the future to not only treat atrial fibrillation, myocardial ischemia/reperfusion injury, but also diabetic atherosclerosis.The clinical drug metformin  and a nu−/− mice; decreasing the elevated triglyceride, total cholesterol, low-density lipoprotein, lipoprotein A, and lipoprotein B and increasing the reduced high-density lipoprotein. The effects of acacetin on the lipid profile might contribute to the reduction of atherosclerotic process. One of limitations was that characterization  of liver and adipose tissues and the related mechanisms of lipid regulation by acacetin were not explored in detail, which remain to be clarified in the future. Second limitation was that Sirt1 and Sirt3 activities in response to acacetin was not evaluated to demonstrate how NAD+ increases Sirt1 and/or Sirt3 activity by changing the deacetylase capacity. Third limitation was that HUVECs were used as a cell model in the present study, which may not be a perfect cell model for correlating atherosclerosis. Fourth limitation was that we did not use D-mannitol to serve as osmotic control in HUVECs, since no effect of D-mannitol (27.5 mM with 5.5 mM glucose) on cell viability was observed in the preliminary experiment, which is similar to the observation from the recent report  across the Taiwan Straits of the National Natural Science Foundation of China.G-RL was employed in part by Nanjing Amazigh Pharma Limited, Nanjing, China.The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


BRAF V600E targetable mutation by digital-PCR from cell-free-DNA and EV-derived DNA (ctDNA) in serum, plasma and cerebrospinal fluid (CSF) isolated from a cohort of 29 CNS pediatric patients. Here we demonstrate that ctDNA isolated from serum and plasma could be successfully analyzed to obtain tumor genetic information which could be used to guide critical treatment decisions.Pediatric Central Nervous System (CNS) tumors are the most fatal cancer diseases in childhood. Due to their localization and infiltrative nature, some tumor resections or biopsies are not feasible. In those cases, the use of minimally invasive methods as diagnostic, molecular marker detection, prognostic or monitoring therapies are emerging. The analysis of liquid biopsies which contain genetic information from the tumor has been much more widely explored in adults than in children. We compare the detection of  Central Nervous System (CNS) tumors are the most common solid tumors and the most deadly forms of cancer in childhood , with anBRAF gene point mutation in the exon 15 at codon 600 resulting in an aminoacid replacement from valine to glutamic acid (BRAF p.V600E) [BRAF V600E mutation presence in pediatric Low Grade Gliomas is that the 10-year progression-free survival rate decreases in almost 40% [The main advantage of liquid biopsy application in cancer disease is that it better reflects the biology of the entire tumor and the intra-tumor heterogeneity, guiding for an appropriate therapy or disease monitoring along tumor progression . In thisp.V600E) . This mup.V600E) . The relmost 40% , howevermost 40% ,20,21. Amost 40% .One of the most relevant challenges in clinical application of liquid biopsies is the development of methods that could detect low-allele-fraction variants . Thus, dBRAF V600E in both liquid biopsy sources. Its clinical application could be used to guide critical treatment decisions in those pediatric patients and other pathologies in which that mutation is quite predominant such as melanoma, colorectal carcinoma or papillary thyroid carcinoma [In the present study we have isolated ctDNA, including EV-derived DNA, from a cohort of 29 pediatric cancer patients diagnosed with CNS tumors from serum, plasma and CSF. We show that ctDNA found in serum and plasma could provide the genetic characteristics of the original tumor. More interestingly from a therapeutic point of view, we have identified the actionable driver mutation arcinoma .To explore the possibility of using ctDNA obtained from liquid biopsies as a biomarker source, we isolated serum, plasma and CSF from 29 pediatric patients. As lumbar puncture is not a risk-free invasive procedure, our CSF samples were only obtained from those patients in which its isolation was required for another medical procedure, such as the study of tumor cell dissemination or to explore CNS infections. Moreover, we sequenced DNA obtained from leukocyte suspension to discard germline mutations .BRAF gene and study its V600E mutation. From our cohort, 31.03% of the patients underwent biopsy  and used it to calculate the expected false positive calls (ΛFP) for each sample. Finally, we calculated each p value as proposed by Milbury et al.  and usedBRAF sequence in ctDNA from the serum of all patients involved in the study, except for LI.18, which was not available (N = 28). We detected the BRAF V600E mutation in three patients . Then, we studied all plasma samples (N = 29) and identified the mutation in three thalamic astrocytoma patients . Unfortunately, no amplification was detected in 38.46% of ctDNA isolated from CSF, in which only two samples (LI.4 and LI.37) showed the presence of the mutation  [BRAF V600E was confirmed by dPCR in serum or plasma. Although recent studies show that CSF is more representative of the original tumor genetic features than plasma in Glioblastoma patients [BRAF mutation. Other groups reported this similar limitation with ctDNA amplification problems in 33% of CSF samples depending on the CSF obtention procedure [In the present study, we isolated cfDNA and EV-derived DNA from different types of liquid biopsies. Serum, plasma and CSF were obtained simultaneously from a cohort of pediatric patients N = 29) diagnosed with CNS tumors, avoiding bias produced by the collected time . We evalas (PXA) , as patias (PXA) , which c diagnosepatients , we cannrocedure .To the best of our knowledge this is the first study to compare different sources of liquid biopsies in pediatric cancers , an unmeBRAF V600E sensitivity values are slightly lower than those observed by other authors in other pathologies [Our hologies . One poshologies , so ctDNhologies . Neverthhologies , as has hologies , in whichologies .BRAF V600E mutation analysis in cfDNA and EV-derived DNA found in serum could identify a subset of patients that could respond to certain target inhibitors, as we observed in the patient LI.31, improving the overall survival and progression-free survival. Moreover, it could help identify resistance mechanisms during therapy or monitor disease progression or relapse. In those patients, resistance occurs with the reestablishment of MAPK pathway or by the activation of PI3K/Akt signaling [ignaling . In some CNS tumors first biopsies or follow up re-biopsies are often unfeasible, so the analysis of liquid biopsies could overcome these difficulties and improve treatment personalization by screening for point mutations or measuring tumor burden .Liquid biopsies including plasma, serum and CSF were obtained from 29 patients diagnosed with CNS tumors. CSF samples were collected by a lumbar puncture  or from an external ventricular device . Plasma and serum samples were obtained from peripheral blood samples by centrifugation. Buffy coats were isolated by Ficoll™ density gradient. Plasma, serum, buffy coat and CSF were stored at −80 °C. 500 µL of plasma, serum and CSF-derived circulating DNA was extracted with the QIAamp Circulating Nucleic Acid Kit , as per the manufacturer’s protocol. Then, DNA concentration was determined using Qubit™ dsDNA High Sensitivity assay kit in a Qubit™ 3.0 fluorometer  according to the manufacturer’s proceedings. DNeasy Blood and Tissue Kit (Qiagen) was used to obtain germline DNA.2O, QuantStudio™ 3D Digital PCR Master Mix, and the ready-to-order TaqMan Assays (20X), ID: AH6R5PH for (BRAF) ). Results were analyzed with QuantStudio™ 3D Analysis Suite Cloud Software (ThermoFisher Scientific) and the mean number of copies per µL was calculated.The digital PCR (dPCR) was performed according to the reference protocol for rare mutation using QuantStudio™ 3D Digital PCR System (ThermoFisher Scientific). We combined ctDNA with nuclease-free HFor conventional PCR assays gDNA was amplified using these nucleotide sequences of the specific primers: Fw 5′CATGAAGACCTCACAGTAAA 3′ and Rv: 5′AGACAACTGTTCAAACTGAT 3′. PCR reactions were performed following the protocol of the Paq5000 enzyme . Thermal cycling conditions consisted of a 10 min denaturation step at 95 °C followed by 40 cycles at 95 °C for 20 s, 53.5 °C for 20 s and 72 °C for 30 s. Final PCR products were electrophoretically separated on 1.8% agarose gels.® Terminator v3.1 Cycle Sequencing Kit (ThermoFisher Scientific). Capillary electrophoresis was performed on a 3130 Genetic Analyzer . Sequencing reaction products were analysed with the Sequencing Analysis software (Applied Biosystems) and aligned using BLAST algorithm.Sequencing reactions were performed with BigDyePatient LI.31 underwent brain Magnetic Resonance Imaging at either 1.5 or 3 T on different magnets and using different protocols during the 5-year period of follow-up. Follow-up brain MRI for assessment of response was performed no less frequently than every 3 months. The MR imaging protocol used currently for brain tumor in our institution includes at least the sequences: axial T2 TSE/FSE 3 mm, axial Flair TSE/FSE 3–4 mm, diffusion weighted images , 3D T1 MPRAGE/SPGR/FFE/TFE 1–1.5 mm with and without gadolinium contrast administration.Samples from patients were kindly provided by the Pediatric Hematology and Oncology Unit-HM Hospitales . Samples were processed following current procedures and frozen immediately after their reception. All patients participating in the study signed respective informed consent, and protocols were approved by institutional ethical committees .BRAF V600E mutation by dPCR we obtained the average ratio of false positives from negative control samples, RFP = 1.93 × 10−4. This ratio was used to obtain the average false positives, ΛFP, expected for each sample. Finally, a Poisson distribution with mean ΛFP was used to obtain the significance of the observed positive calls for each sample. The null hypothesis that observed positive calls are false positives was rejected with a confidence threshold of 95%.To assess the significance of the detection of BRAF V600E by dPCR detection in liquid biopsy from CNS pediatric cancers. Therefore, a robust validation with samples from a large cohort is required to turn it into a validated clinical tool.One of the main challenges in clinical practice is to ensure high accuracy for biomarker detection. Although further studies increasing the number of CSF derived ctDNA are required, we found serum as the most promising alternative rather than plasma for