Cell-free circulating tumour DNA (ctDNA) in plasma has been shown to be informative of the genomic alterations present in tumours and has been used to monitor tumour progression and response to treatments. However, patients with brain tumours do not present with or present with low amounts of ctDNA in plasma precluding the genomic characterization of brain cancer through plasma ctDNA. Here we show that ctDNA derived from central nervous system tumours is more abundantly present in the cerebrospinal fluid (CSF) than in plasma. Massively parallel sequencing of CSF ctDNA more comprehensively characterizes the genomic alterations of brain tumours than plasma, allowing the identification of actionable brain tumour somatic mutations. We show that CSF ctDNA levels longitudinally fluctuate in time and follow the changes in brain tumour burden providing biomarkers to monitor brain malignancies. Moreover, CSF ctDNA is shown to facilitate and complement the diagnosis of leptomeningeal carcinomatosis. DNA circulating in the plasma of cancer patients carries features of the primary tumour, however such DNA is found in low levels in brain cancer patients. Here, the authors show that circulating tumour DNA can be detected in the cerebral spinal fluid of cancer patients and that this better recapitulates the primary tumour compared to DNA from the plasma. The genomic characterization of tumours is crucial for the optimal diagnosis and treatment of cancer. Given the reported spatial and temporal intratumour heterogeneity, repeated biopsies are required for an adequate characterization of the somatic genetic alterations found in human cancers1456The cerebrospinal fluid (CSF) is in intimate contact with tumour cells in central nervous system (CNS) cancers and, recently, ctDNA has been shown to be present in the CSF of patients with brain tumours8To study and compare the ctDNA present in the CSF with plasma ctDNA, we sequenced DNA obtained from tumour samples, germline DNA , plasma and CSF of a cohort of 12 patients  with CNS restricted disease and barely any visceral tumour burden to facilitate the comparison of the contribution of the brain tumour DNA into the CSF or plasma ctDNA. In all cases, CSF ctDNA was detected and harboured gene mutations that were either absent or detected with lower MAFs in plasma ctDNA .We next sought to determine whether CSF ctDNA would be more representative of the brain lesions than plasma ctDNA. To this end we divided the patients into two groups depending on the amount of extracranial tumour burden .Importantly, in patients with a CNS restricted disease , the MAFPIK3CB M819L, PIK3CB Q818H, AHNAK2 L5292V) exclusively present in the meningeal lesion. The gene mutations with the highest MAFs of the brain metastasis and the private mutations in the meningeal lesions were present in the CSF ctDNA and not in the plasma ctDNA  at sequential time points , Fig. 3.The identification of CSF ctDNA led us to the hypothesis that cell-free DNA in the CSF could be used as a diagnostic tool for LC. The diagnosis of LC relies on the detection of malignant cells in the CSF of patients with clinical symptoms. Diagnosis of LC is not trivial and its misdiagnosis has important clinical implications. To define whether the analysis of CSF ctDNA can be employed to enhance the sensitivity of the detection of LC by cytopathologic analysis of CSF, we performed standard of care cytopathologic analysis and CSF ctDNA sequencing in the same samples obtained from three breast cancer patients with clinical signs and symptoms suggestive of LC.Importantly, there were discrepancies between the cytology and our CSF ctDNA analysis . In BMBCEGFR, PTEN, ESR1, IDH1, ERBB2, FGFR2) can be identified. We observed that CSF ctDNA has a significantly higher sensitivity than plasma for CNS genomic alterations and can be used to detect brain tumour private mutations and to monitor brain tumour progression. In addition, we provided evidence that the analysis of CSF ctDNA may complement the diagnosis of LC.In this study, we identified and characterized ctDNA in the CSF of patients with brain lesions and compared it with plasma ctDNA. We showed that CSF ctDNA is more representative of brain tumour genomic alterations than plasma and putative actionable gene mutations and CNA  Warm Autopsy Program. Patients with breast cancer and lung cancer with brain metastasis, and GBM and medulloblastoma were enrolled as part of VHIO Prospective Translational Program, which studies plasma and CSF-derived biomarkers. Patients with lung cancer with brain metastasis were enrolled as part a collaborative effort with Dexeus University Hospital  and the research was approved by the local institutional review board (IRB)/ethics committee of both hospitals. VHIO Warm Autopsy Program and the Prospective Translational Program were approved by the IRB of Vall d'Hebron University Hospital . Informed consent was obtained from all patients.The diagnosis of each metastatic lesion was confirmed on review of routine hematoxylin and eosin-stained slidesDNA samples from CNS tumours (primary brain tumours or CNS metastases) of 23 cases, non-CNS metastases, CSF and plasma samples as well as germline DNA were subjected to targeted capture massively parallel sequencing at the Memorial Sloan Kettering Cancer Center Integrated Genomics Operation (iGO), using the Integrated Mutation Profiling of Actionable Cancer Targets (MSK-IMPACT) platformTargeted sequencing was performed as previously described617Paired-end 100-bp reads were generated on the Illumina HiSeq2000 , and reads were aligned to the reference human genome hg19 using the Burrows-Wheeler AlignerDNA (500ng) extracted from brain tumour and germline samples from GBM1, GBM2 and GBM3, BMBC1, BMLC1 and BMLC2 cases were subjected to exome sequencing. An average of 100 million 100-bp paired-end reads were generated for each sample, equivalent to an average depth of 260 × (range of 190–315 × ). Exome sequencing was performed using the Nextera Rapid Capture Exome kit  on an Illumina HiSeq 2000 instrument using a validated protocolddPCR of plasma and CSF were performed using the QX200 Droplet Digital PCR system (Bio-Rad) according to manufacturer's protocols and the literaturePhylogenetic trees were constructed using the maximum parsimony method. The trunks of the trees were rooted by a germline DNA sequence that did not have any of the somatic mutations. Trunk, branch and sub-branches lengths are proportional to the number of mutations.A Mann–Whitney test was performed for statistical analysis. Data in graphs are presented as means±s.d.Accession codes: Whole-exome and targeted capture massively parallel sequencing have been deposited in the Sequence Read Archive (SRA) under accession code SRP049647.How to cite this article: De Mattos-Arruda, L. et al. Cerebrospinal fluid-derived circulating tumour DNA better represents the genomic alterations of brain tumours than plasma. Nat. Commun. 6:8839 doi: 10.1038/ncomms9839 (2015).Supplementary Figures 1-4 and Supplementary Tables 1-8List of the customized breast cancer and MSK-IMPACT platforms (254 breast cancer-associated genes and 341 cancer-associated genes) involved in the targeted capture massively parallel sequencing employed in this study. The lists of 488 unique cancer-associated genes derived from both platforms and genes in common to both platforms are also provided.Somatic single nucleotide variants (SNVs) and insertions and deletions (indels) from the IMPACT platform present in patients with glioblastoma, brain metastasis from lung cancer (BMLC) and brain metastasis from breast cancer (BMBC) subjected to warm autopsies. SNVs and indels from the customized breast cancer present in four BMBC (cases 1-4) subjected to warm autopsies.Sequencing metrics for the MSK-IMPACT and breast panels.


P = 0.54). One-year relapse-free survival was 80.7% in PO compared to 87.3% in IV patients, all-time adjusted HR 3.8 . During the first 12 months, neutropenia of ≤ 0.5 × 109/L occurred in 9 (16%) PO and 0 (0%) IV cyclophosphamide patients (P = 0.003). The number of patients admitted with one or more infections was 16 (28%) in the PO group and 9 (16%) in the IV group, adjusted OR 2.2 . We observed an increased risk of neutropenia, a trend towards increased risk of death and an admission with infection with PO cyclophosphamide. This adds certainty to previous studies, indicating that PO administration induces greater marrow toxicity. Infection-related admissions within 12 months of starting cyclophosphamide were higher than those in clinical trials, possibly reflecting the unselected nature of this cohort.We aimed to compare risk of death, relapse, neutropenia and infection requiring hospital admission between unselected ANCA-associated vasculitis (AAV) patients according to whether cyclophosphamide induction was by daily oral (PO) or pulse intravenous (IV) route. We identified all newly diagnosed AAV patients treated with PO or IV cyclophosphamide between March 2007 and June 2013. We used Cox and logistic regression models to compare mortality, relapse and adverse events and adjusted these for age, renal function and other significant confounders. Fifty-seven patients received PO and 57 received IV cyclophosphamide. One-year survival was 86.0% in PO and 98.2% in IV patients; all-time adjusted hazard ratio (HR) for PO compared to that of IV cyclophosphamide was 1.8 (95% CI 0.3–10.6,  Anti-neutrophil cytoplasmic antibody (ANCA)-associated vasculitis (AAV) is the commonest life-threatening vasculitis of adults , and is n = 149) comparing the two regimens, demonstrated that both treatment routes were equally efficacious. Pulsed IV therapy had fewer side effects including leukopenia but is reported to have a higher relapse rate than the continuous oral route; however, long-term follow-up showed no difference in mortality or renal function at 5 years with either route  2.8) years in the PO and 5.3 (SD 2.6) years in the IV group.Baseline characteristics and clinical features were significantly different between the two groups Table , reflectPrednisolone dose at diagnosis was median 45 mg (IQR 30–60 mg) in the PO cyclophosphamide group and 60 mg (42.5—60 mg) in the IV cyclophosphamide group. By 1 year, both groups had median prednisolone dose of 5 mg (Table Results are shown in Table P = 0.47).Overall, 22 patients died in 553 patient-years of follow-up . One-year survival was 86.0% in PO and 98.2% in IV patients. Survival to the end of the follow-up period was 70% in PO and 91% in IV patients. Unadjusted hazard ratio (HR) for the whole follow-up period for PO compared to IV cyclophosphamide was 3.9 (95% CI 1.4–10.4), and adjusted HR was 1.9 .One-year relapse rate was 4 (7%) in PO and 6 (11%) in IV patients; relapse rate to the end of the follow-up period was 14 (25%) in PO and 24 (42%) in IV patients. Unadjusted hazard ratio (HR) for the whole follow-up period for PO compared to that of IV cyclophosphamide was 0.5 (95% CI 0.3–1.0), and adjusted HR was 1.0  PO and 0 (0%) IV patients (P = 0.003). It was not possible to calculate an odds ratio because the outcome did not occur in IV patients. The lowest documented neutrophil count was 0.1 in the PO group and 0.94 in the IV group. Five patients in the PO group were treated for neutropenic sepsis with no recorded cases in the IV group.During the first 12 months after initiating treatment, documented neutropenia of ≤ 0.5 × 10P = 0.24).Overall, 25 (22%) patients were admitted to hospital with ≥ 1 infection during 12 months after initiation treatment: 16 (28%) with PO and 9 (16%) with IV cyclophosphamide. Unadjusted OR for admission with infection in PO compared to that of IV cyclophosphamide was 2.0 (95% CI 0.8–5.0), and adjusted OR was 2.2 ; incident diabetes, in 14 (13%); VTE, in 8 (7%); MI, in 8 (7%); stroke, in 8 (7%) and any cancer, in 9 (8%).E. coli sepsis), 1 progressive lung fibrosis, 1 lung cancer, 1 retroperitoneal haemorrhage and 1 unknown. Of the 13 deaths after the first year, 7 (54%) were due to infection  1 MI, 1 old age, 1 renal failure and 3 unknown.Cause of death is recorded for 18 of the 22 deaths. Of the 9 deaths in the first year, 5 (56%) were due to infection  and that data capture-recapture techniques suggested our case identification technique resulted in near complete case capture , resultiOur study is limited by its observational nature. However, observational studies of outcome are important in rare diseases and can help to increase the level of certainty about specific clinical questions. The main limitation is the risk of confounding; treatment route was likely to have been determined by the baseline characteristics, with older patients and those with renal dysfunction (both risk factors for poor outcomes) more likely to be treated by nephrology and therefore receive PO cyclophosphamide. The sample size was too small to use propensity score methods to adjust for this; we therefore adjusted the analysis for the known confounders . We are unlikely to have been completely successful in adjusting; however, despite the theoretical limitations, our adjustment led to attenuation of the crude differences in treatment effects in the adjusted models.Whereas in the CYCLOPS study, patients received a median cyclophosphamide dose of 8.58 g IV and 18.05 g PO  and our Contrasting with CYCLOPS, we observed an overall rate of admission with infection during the first year of 22% . Only a few admissions were at times of frank neutropenia, and the steroid doses in our cohort were marginally lower than those of the CYCLOPS protocol. The average age of patients in our IV treatment cohort was similar to that of the CYCLOPS cohort; however, our PO cohort was significantly older; advancing age is a risk factor for hospitalisation with infection. Additionally, a quarter of our cohort was made up of patients who would have been excluded by the CYCLOPS trial protocol, e.g. life-threatening disease or creatinine > 500 μmol/L.The finding of differences between patients with AAV presenting to nephrology or rheumatology services is consistent with that of a recent paper from Ireland describing patients with AAV presenting to Galway University Hospitals from June 2002 to July 2011 . They reThe main cause of mortality in our study was infection, accounting for 56% of deaths in the first year after diagnosis and 54% of later deaths, with none recorded as due to active vasculitis, suggesting that in our cohort, the increased risk of infection extends beyond the first year. This does not completely align with previous studies of cause of death where in the first year after diagnosis, the most frequent causes were infection (32–48% of deaths) then active vasculitis (18–19% of deaths), with later deaths most frequently due to cardiovascular disease, malignancy and renal failure , 17.In conclusion, PO cyclophosphamide was associated with an increased risk of neutropenia, and a trend towards increased risk of death, and admission with infection with PO cyclophosphamide. This adds to the level of certainty that PO cyclophosphamide is associated with greater marrow toxicity that was suggested in the CYCLOPS study results and a previous meta-analysis that demonstrated increased risk of leukopenia with this route . This isOf note in our study, in the setting of routine clinic practice, over 20% of the patients were admitted with infection in the first year. It remains to be seen if further developments in the management of ANCA vasculitis (e.g. steroid sparing protocols) can impact upon this, but it highlights the need for ongoing clinical vigilance during the treatment of this complex disease.


The multivariate-adjusted values of partial correlation coefficients for these inflammation-related markers were −0.098, −0.079, −0.114, −0.090, and −0.079 , respectively. Among the individual carotenoids, those with the most predominant association were lutein-zeaxanthin and total carotenoids for retinol-adjusted RBP4 and RBP4, alpha- and beta-carotene for hsCRP, and alpha-carotene for MCP1 and TNF-alpha. No significant associations were observed for IL-6 and IL-1beta. (4) Conclusions: Serum carotenoids were inversely associated with RBP4, hsCRP, MCP1 and TNF-alpha among middle-aged and elderly Chinese adults.(1) Background: Carotenoids may be inversely associated with inflammatory markers . However, data are scarce on retinol binding protein 4 (RBP4) in humans. We examined the associations among serum carotenoids, RBP4 and several inflammatory markers in a Chinese population. (2) Methods: This community-based cross-sectional study included 3031 participants  aged 40–75 years in Guangzhou, China. Serum concentrations of carotenoids, RBP4, and inflammatory markers were measured. (3) Results: Generally, serum individual and total carotenoids were significantly and inversely associated with retinol-adjusted RBP4, RBP4, hsCRP, MCP1, and TNF-alpha levels. Age- and gender-adjusted partial correlation coefficients between total carotenoids and the above inflammatory markers were −0.129, −0.097, −0.159, −0.079, and −0.014 (all  Inflammation is an essential component of immunosurveillance and host defense. However, a chronic increase in inflammatory markers, such as interleukin (IL)-6, IL-1, tumor necrosis factor α (TNF-α) ,3, playsFruits and vegetables are rich in carotenoids, a group of fat-soluble phytochemicals. The levels of blood carotenoids are inversely associated with risks of many chronic diseases, including T2DM ,8,9, CVDRetinol binding protein 4 (RBP4) is the sole retinol transporter in the blood and is secreted from the liver and adipocytes   with RBP4 and hsCRP, a traditional inflammatory marker, and examined the associations with a variety of other inflammatory markers including MCP1, IL-1, IL-6, and TNF-α, in a middle-aged and older Chinese population.www.clinicaltrials.gov as NCT03179657. The study protocol was approved by the Ethics Committee at Sun Yat-sen University.Participants included in this community-based cross-sectional study were from The Guangzhou Nutrition and Health Study (GNHS); the study enrolled 3169 participants from 2008 to 2010, and 832 new subjects were recruited in 2013 in Guangzhou, China. Participants with cancer, CVD, stroke and mental illnesses were excluded. Furthermore, participants with missing carotenoid and inflammatory markers were excluded. Finally, 2925 participants with serum carotenoid data and RBP4 or at least one inflammatory marker were included in this study. Written informed consent was obtained from all of the subjects. This study has been registered at 2 (in kg/m2). The waist circumference (WC) was also measured.The following information from the participants was obtained: socio-demographic characteristics, lifestyle factors, smoking and drinking habits, and multivitamin and calcium supplementation; the data were collected using a face-to-face interview with a structured questionnaire. The metabolic equivalent (MET) was calculated from a 19-item physical activity questionnaire . Height g for 15 min at 4 °C, and the samples were stored at −80 °C until further analysis. Serum total cholesterol (TC) levels were measured by colorimetric methods using commercial kits  in a Hitachi 7600-010 automated analyzer .Venous blood samples were collected after an overnight fast. Serum was separated by centrifugation at 1500× v/v), and phase B contained acetonitrile-methanol-tetrahydrofuran-ammonium acetate 55:35:5:5, v/v. The Waters 2998 diode-array detector was used . Then, the chromatograms of serum carotenoids was shown in Reversed-phase high-performance liquid chromatography (HPLC) was used to simultaneously measure the serum levels of α-carotene, β-carotene, β-cryptoxanthin, lycopene and lutein/zeaxanthin with the methods described by Burri et al.  and withSerum RBP4 levels were measured using the enzyme-linked immunosorbent assay (ELISA) kit , and the absorbance value was determined by a microplate spectrophotometer . The lowest level of RBP4 that can be detected by this assay is 380 pg/mL (between-plate CV: 3.59%). High sensitivity C-reactive protein (hsCRP) was measured using the Cardiac C-Reactive Protein (Latex) High Sensitive (CRPHS) kit in the cobas c701 autoanalyzer according to the instructions by the manufacturer (inter-assay CV: 2.07%).Serum levels of monocyte chemoattractant protein-1 (MCP1), tumor necrosis factor (TNF-α), Interleukin-6 (IL-6), Interleukin-1β (IL-1β) were measured in approximately 1/3 of the total subjects selected from one of three batches of serum samples according to the batch number sequence. Human FlowCytomix  and the Human Basic Kit FlowCytomix  on a BD FACSCalibur instrument  were used for the measurements . The bett-test or Mann-Whitney U test was used to compare the characteristics of the participants. For the categorical variables, the data were presented as frequencies (percent) and were compared using Chi-square test.Continuous variables were presented as the means ± standard deviation for normally distributed data or the median and the 25th–75th percentile ranges for data with a skewed distribution. The Logarithmic transformed data of the skewed distribution variables  and their Z-scores were then used in further correlation analyses. Since serum RBP4 levels are positively correlated with retinol levels, in this study, we also used crude RBP4 and retinal-adjusted RBP4 (aRBP4) as was done in a previous study .Partial correlation analyses were used to assess the associations of the concentrations of total or each individual carotenoid with RBP4, retinol-adjusted RBP4, and inflammatory markers. The first model was adjusted for age and gender. Multiple models were further adjusted for BMI, TC, marital status, education level, household income (Yuan/month per person), smoking and drinking habits, vitamin supplements, and physical activity in all subjects and in women and men separately.p < 0.05 was considered statistically significant. All of the analyses were conducted using SPSS16.0 software .Further, concerning the biological relevance, we used logistic regression model to determine the association between serum concentrations of carotenoids and elevated inflammation levels which determined by hsCRP (≥1.0 mg/L)  and RBP4p values < 0.05).The characteristics of the study participants are shown in r = −0.140 and −0.120) and RBP4 (r = −0.093 and −0.084), α-carotene and β-carotene for hsCRP , and α-carotene for MCP1 (r = −0.088 and −0.090) and TNF-α (r = −0.080 and −0.096) in Models 1 and 2 .The associations between serum values of carotenoids and RBP4 or inflammatory markers are shown in r = −0.091, p < 0.01) and lycopene  was observed in women. The correlation between carotenoids and hsCRP tended to be similar in women and men. Furthermore, we adjusted for medications , which could affect the concentration of inflammatory markers; the associations that were examined were slightly attenuated  and from 0.44 to 0.75 for RBP4  (We further explored the associations between serum levels of carotenoids and elevated hsCRP and RBP4. Multivariate-adjusted ORs (95% CI) of elevated hsCRP and RBP4 for each unit (log μmol/L) increase in total carotenoids were 0.42 (0.29–0.61) for hsCRP and 0.43 (0.27–0.67) for RBP4. For individual carotenoids, the corresponding ORs ranged from 0.48 to 0.76 for hsCRP  .In this community-based cross-sectional sample of 2925 middle-aged and older women and men, we first found that serum carotenoid concentrations were inversely associated with retinol-adjusted RBP4, RBP4, hsCRP, MCP1 and TNF-α in general adults.RBP4 plays a causal role in insulin resistance by reducing immunity and inflammatory mechanisms in adipose and vascular tissues ,25,39. RThe mechanisms underlying the favorable association between carotenoids and RBP4 remain unclear. An in vitro study showed that the antioxidant property of resveratrol decreases the cellular mRNA levels of RBP4 and the RBP4 concentration in the culture medium; resveratrol also reduces triacylglycerol content and the mRNA levels of lipogenic genes that are associated with the over-production of RBP4 . The dowIn this study, we detected the strongest inverse association between serum carotenoids and hsCRP among the inflammatory markers in all subjects and observed similar but relatively less pronounced associations with MCP1, TNF-α and IL-1β (but not IL-6) in a subset of samples. In line with our findings, the favorable associations between serum carotenoids and inflammatory markers were observed in several cross-sectional studies ,45,46,47As summarized by Kaulmann and Bohn , carotenConsistent with many previous results ,53, signThere are several strengths to this study. To our knowledge, this is the first study that reported an inverse association between serum concentrations of five common carotenoids and RBP4 in a relatively large population. Our findings provided clues for the prevention of insulin resistance and inflammation-related chronic diseases via the use of carotenoid rich-foods in the diet aimed at lowering serum RBP4 levels. Next, we explored the associations of five types of carotenoids, RBP4 and five inflammatory markers and provided systematic evaluations, avoided false negative results, and found similar results among different carotenoids to reduce the probability of false positive results. Our study has several potential limitations. First, we could not examine a cause-and-effect relationship due to the observational study design. Second, the ELISA method used to determine RBP4 in our study measured the total RBP4 concentration and could not differentiate between holo-RBP4 (bound to retinol) and apo-RBP4 (not bound to retinol); in addition, we were unable to distinguish between the full-length and the truncated forms of RBP4 . Third, In conclusion, our findings show that serum carotenoid concentrations are inversely associated with RBP4 and other inflammatory markers  in middle-aged and elderly Chinese men and women. Considering the pitfalls of cross-sectional studies, further experimental studies are needed to clarify the effects of carotenoids on levels of serum RBP4 and other inflammatory markers.


Hyperhomocysteinemia is a risk factor for cognitive decline and dementia, including Alzheimer disease (AD). Homocysteine (Hcy) is a sulfur-containing amino acid and metabolite of the methionine pathway. The interrelated methionine, purine, and thymidylate cycles constitute the one-carbon metabolism that plays a critical role in the synthesis of DNA, neurotransmitters, phospholipids, and myelin. In this study, we tested the hypothesis that one-carbon metabolites beyond Hcy are relevant to cognitive function and cerebrospinal fluid (CSF) measures of AD pathology in older adults.n = 72) or without (n = 48) cognitive impairment. Liquid chromatography-mass spectrometry was performed to quantify one-carbon metabolites and their cofactors. Least absolute shrinkage and selection operator (LASSO) regression was initially applied to clinical and biomarker measures that generate the highest diagnostic accuracy of a priori-defined cognitive impairment  and AD pathology  to establish a reference benchmark. Two other LASSO-determined models were generated that included the one-carbon metabolites in CSF and then plasma. Correlations of CSF and plasma one-carbon metabolites with CSF amyloid and tau were explored. LASSO-determined models were stratified by apolipoprotein E (APOE) ε4 carrier status.Cross-sectional analysis was performed on matched CSF and plasma collected from 120 older community-dwelling adults with , S-adenosylmethionine (SAM), serine, cysteine, and 5-methyltetrahydrofolate (5-MTHF) improved the diagnostic accuracy to 87.4%. A second model derived from plasma included cystathionine, glycine, methionine, SAH, SAM, serine, cysteine, and Hcy and reached a diagnostic accuracy of 87.5%. CSF SAH and 5-MTHF were associated with CSF tau and p-tau181. Plasma one-carbon metabolites were able to diagnose subjects with a positive CSF profile of AD pathology in APOE ε4 carriers.The diagnostic accuracy of cognitive impairment for the reference model was 80.8% and included age, years of education, AβAPOE ε4 carriers.We observed significant improvements in the prediction of cognitive impairment by adding one-carbon metabolites. This is partially explained by associations with CSF tau and p-tau181, suggesting a role for one-carbon metabolism in the aggregation of tau and neuronal injury. These metabolites may be particularly critical in The online version of this article (doi:10.1186/s13195-017-0270-x) contains supplementary material, which is available to authorized users. MTHFR) and environmental factors influence one-carbon metabolism; however, genetic factors are thought to play a minor role, whereas dietary intake of folate, vitamin B6, and vitamin B12 explains 35% of the variation [The methionine, purine, and thymidylate cycles together encompass one-carbon metabolism in the cytosol . Both ge6, B12, and folate, as well as oxidative damage [S-adenosylmethionine (SAM), S-adenosyl-L-homocysteine (SAH), and 5-methyltetrahydrofolate (5-MTHF) balance may operate on several mechanisms that support cognitive function, ranging from epigenetic modulation of synaptic function in the hippocampus to methylation reactions in the liver that convert phosphatidylethanolamine to phosphatidylcholine and facilitate the delivery of essential fatty acids  to the plasma and the brain [The most-studied biomarker of one-carbon metabolism is homocysteine (Hcy), a sulfur-containing amino acid generated by the metabolism of methionine . Inherite damage , 12. Howe damage . There mhe brain –18.APOE) gene is a well-known and major genetic risk factor for AD [APOE genotype and environmental factors, such as diet, on the risk of dementia and AD [APOE status, and the association between B12 and cognitive function was indeed shown to be stronger in APOE ε4 carriers [12 and Hcy on gray matter volume may also be influenced by APOE genotype in AD [APOE remains to be more broadly investigated.The ε4 allele of apolipoprotein E . Cognitively intact participants were recruited from the community through advertisement or among the spouses of memory clinic patientsVenous and lumbar punctures were performed between 8:30 and 9:30 a.m. after overnight fasting. For lumbar puncture, a standardized technique with a 22-gauge “atraumatic” spinal needle and the subject in a sitting or lying position was applied . A volumThe measurements were performed using commercially available enzyme-linked immunosorbent assay kits and TaqMan assays  as described in Additional file see Additional file Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was performed to measure absolute concentrations of metabolites and cofactors of one-carbon metabolism in CSF and blood plasma as described previously   or dementia was based on neuropsychological and clinical evaluation and made by a consensus conference of psychiatrists and/or neurologists and neuropsychologists prior to subject inclusion. The participants had no major psychiatric or neurological disorders, nor did they have substance abuse or severe or unstable physical illness that might contribute to cognitive impairment. Magnetic resonance imaging and computed tomographic scans were used to exclude cerebral pathologies possibly interfering with the cognitive performance. Subjects known to take folate and B-vitamin supplementation were excluded from the study. MCI was diagnosed according to widely used consensus recommendations . Subjectmer type  with a C1–42) ratio: “low” when p-tau181/Aβ1–42 was ≤0.0779 or “high” when p-tau181/Aβ1–42 was >0.0779, considered as negative and positive CSF profiles of AD pathology, respectively. This threshold optimized the Youden index [Subjects were also classified into two groups on the basis of their CSF tau phosphorylated at threonine 181 (p-tau181)/β-amyloid 1–42 peptide chain  logistic regression , we seleage in R , which aage in R . Resultsage in R  and accu1–42, tau, and p-tau181 were performed with Pearson’s statistics and Bonferroni-corrected for multiple comparisons. Significant correlations between the metabolites and cofactors (p < 0.05) with CSF tau or p-tau181 were studied further in a linear regression model with tau or p-tau181 as the dependent variable and the following explanatory covariates: each metabolite, age, sex, APOE genotype , CDR (0 versus >0), and CSF albumin index (only for plasma metabolites). Values of the regression terms were reported, and their differences from 0 were assessed with t tests. Interaction terms between the metabolites and age, sex, CDR, or APOE genotype were identified in a type II analysis of variance (ANOVA) with an F test.Two-sided correlation analyses between metabolites and CSF AβAPOE ε4 genotype than the cognitively intact group (CDR 0)  Table . CSF Aβ11–42, CSF tau, and CSF p-tau181 and produced a diagnostic accuracy of 80.8% and an ROC AUC of 0.86 (95% CI 0.79–0.92). This model classified 59 of 72 cognitively impaired subjects correctly and 38 of 48 cognitively intact subjects correctly. By comparison, a majority class prediction [The reference model for classification of cognitive impairment included age, years of education, CSF Aβediction  producedp value of 0.0704 compared with reference model), and the ROC AUC reached 0.92 (CI 0.87–0.96)  and choline (p = 0.01) elevation in the cognitively impaired patients.To evaluate the individual contribution of each metabolite selected in the best models, we conducted group comparisons for each metabolite. CSF methionine and serine were higher in cognitive impairment, with p value of 0.0614), and the ROC AUC was improved to 0.91 (CI 0.86–0.96) . A majority class prediction produced a diagnostic accuracy of about 65% to distinguish subjects with p-tau181/Aβ1–42 ≤ 0.0779 from subjects with p-tau181/Aβ1–42 > 0.0779.The reference model for classification of the CSF profile of AD pathology included age and presence of CSF and plasma one-carbon metabolites were both unable to improve the classification of CSF profile of AD pathology beyond the reference model (data not shown).1–42, tau, and p-tau181 were significantly different between CDR categories . In this subgroup, no correlation was found with CSF levels of Aβ1–42 and individual metabolite levels in both CSF and plasma. Cystathionine, SAH, and 5-MTHF in CSF, as well as Hcy in plasma, showed significant correlations with CSF p-tau181  and an ROC AUC of 0.92 (CI 0.7–1.00) (p = 0.0129 versus the reference model). Seven plasma metabolites were included in this mathematical model  in addition to age, sex, years of education, and CSF albumin index. Box plots of plasma metabolite measurements for non-AD versus AD CSF profiles in APOE ε4 carriers are provided in Additional file The metabolite measurements were considered in a LASSO logistic regression to classify patients with non-AD versus AD CSF profiles in this subgroup of individuals. A reference LASSO logistic regression on known or probable contributing variables  provided a reference model with an accuracy of 83.8% and an ROC AUC of 0.76 (CI 0.55–0.92). Here, a majority class classifier provided 64.9% diagnostic accuracy. Including metabolite markers measured in CSF did not improve the ability to predict an AD biomarker profile compared with the reference model. In contrast, plasma metabolites provided nonsignificant improvement in terms of accuracy but significantly increased performance for AUC Fig. . The modIn the present study, we deployed a LC-MS/MS-based method to expand previous work , targetiOur results highlight the contribution of methionine, serine, choline, and cysteine in addition to the more commonly assessed and reported one-carbon metabolites . Methionine supplementation in wild-type mice can induce neurotoxicity, higher levels of tau phosphorylation and Aβ peptides in the brain, and memory loss . These m1–42 was remarkable and consistent with some studies [1–42 and soluble amyloid precursor protein forms, suggesting a contribution to the accumulation of cerebral amyloid pathology [Several metabolites of the one-carbon cycle measured in CSF and plasma were associated with tau metabolism, reflected by total tau and p-tau181 in the CSF, after controlling for potential confounders. Higher CSF 5-MTHF appeared protective, whereas higher CSF SAH appeared to promote tau aggregation and neuronal injury. These results are consistent with reports demonstrating higher CSF SAH and lower CSF 5-MTHF associated with higher CSF p-tau181 . Accordi studies , 45, but studies , 47. A rathology , making APOE genotype was identified. For example, whereas plasma Hcy appeared insubstantial to CSF tau in the total sample, hyperhomocysteinemia was relevant to CSF tau in the APOE ε4 carriers. This was further corroborated by our prediction of positive AD pathology using the p-tau181/Aβ1–42 ratio after restricting the analysis to APOE ε4 carriers only. Although this interaction between Hcy, APOE, and tau pathology was clear in our analysis, the biological rationale behind this interaction is less clear. A recent report demonstrated that plasma Hcy predicts the conversion from MCI to AD only in APOE ε4 carriers [APOE ε4 carriers and should be further explored.We were unable to predict CSF profiles of AD pathology until an interaction with carriers , adding carriers , may alsTaken together, our results show that one-carbon metabolism is relevant to cognition independent of and beyond AD core pathology. One-carbon metabolism disturbances may cripple DNA repair, methylation, and/or synthesis, as well as reduce the availability of neurotransmitters, phospholipids, and myelin . SeveralThe present study has limitations. We used measures of cognition and CSF measures of AD in a cross-sectional analysis, and the temporality of the associations cannot be extrapolated. Nevertheless, we present the most comprehensive mapping of the one-carbon pathway in both CSF and plasma in older adults. The results are encouraging and in the future should be replicated in an independent cohort as well as confirmed in relation to longitudinal change in cognition and the incidence of AD.Our results suggest that several one-carbon metabolites beyond Hcy are relevant to AD pathology and cognitive function in older adults. This may partially explain why Hcy-lowering trials have been mostly disappointing and opens the possibility that other metabolites in the pathway may also be relevant targets for the prevention of cognitive decline and the underlying neurobiology of AD.


After repeated doses the increase was even more pronounced: hc-DNA increased by about 50 % ). A statistical significant increase in histone levels was detected as early as 4 h after G-CSF injection . In the placebo group no significant changes occurred. Moreover, significantly higher levels of hc-DNA were measured in male compared to female subjects . G-CSF injection substantially increases hc-DNA levels in healthy volunteers. There is a significant gender difference in hc-DNA at the baseline.Granulocyte colony-stimulating factor (G-CSF) is an activator of neutrophil granulocytes. Neutrophil extracellular traps are a defensive mechanism consisting of neutrophils, platelets, DNA, histones and antimicrobial proteins. This study was performed to determine whether G-CSF increases histone-complexed DNA in the plasma of healthy volunteers. In total, 51 healthy volunteers  were treated with G-CSF  and six participants received a placebo. Histone-complexed DNA was measured by enzyme immunoassay in plasma samples at predefined time points . Histone levels were quantified by Western blotting. A single dose of G-CSF rapidly increased hc-DNA by about 50 % ( Granulocyte colony-stimulating factor (G-CSF) not only stimulates the growth and proliferation of hematopoietic progenitor cells, but also activates mature granulocytes. Binding of G-CSF to its receptor prolongs the lifespan of granulocytes, enhances the phagocytic activity and induces degranulation of preformed and stored defensive substances and modulators of the immune system –3. EndogRecently a new defensive mechanism was discovered. In a process called “NETosis” neutrophil extracellular traps (NETs) are formed. In short, stimulation of neutrophils leads to an interaction with the endothelium and to chromatin decondensation by enzymes . Neutrophils then release decondensated DNA, associated antimicrobial factors and histones. These “traps” are ready to catch and attack pathogens . Hence, In a mouse model investigating the prothrombotic state of cancer patients the involvement of G-CSF in NET formation was demonstrated for the first time . Thus, wWritten informed consent was obtained from all study participants before study entry. The study protocol was approved by the Independent Ethics Committee of the Medical University of Vienna, and the trial was registered in a public database . The study was conducted in accordance with the good clinical practice guidelines and the Declaration of Helsinki. The trial took place at the Medical University of Vienna, Department of Clinical Pharmacology, and was conducted between December 8, 2006, and June 17, 2009.Healthy men and women between 18 and 55 years, with a body weight ≤96 kg, were included in the study. All participants signed an informed consent form before any trial-related activity was performed. All female subjects with childbearing potential performed a negative urine pregnancy test before inclusion. All subjects were nonsmokers with normal laboratory values and normal findings in their medical history.Exclusion criteria contained known spleen enlargement, hypersensitivity to any of the formulations’ ingredients, clinically relevant abnormal laboratory values, use of any medication during 2 weeks before the start of the study, a clinically relevant illness within 3 weeks before the start of the study, a recent infection within 1 week, skin abnormalities at the injection site or any other medical condition deemed relevant by the principal investigator.The trial took place at the Department of Clinical Pharmacology of the Medical University of Vienna. The trial was performed to investigate biosimilarity between two G-CSF formulations. In the trial protocol, storage of plasma samples for academic purposes was planned before the initiation of the trial. In one part of the project, healthy volunteers received 300 µg of G-CSF intravenously (i.v.). In another part, a double blind, randomized, active and placebo-controlled, parallel group trial, healthy volunteers were randomized to receive 5 µg/kg G-CSF subcutaneously or a placebo on four consecutive days. This dose was chosen with regard to the primary endpoint of the trials, the absolute neutrophil count. Increasing the G-CSF dose to 10 µg/kg bodyweight does not have a major influence on the absolute neutrophil count , but incg at 4 °C for 15 min. Plasma was stored at −80 °C until analyzed.Samples were drawn using intravenous catheters placed in an appropriate antecubital vein. After discarding the first 2 mm of blood, blood was drawn into sterile blood tubes anticoagulated with 3.8 % sodium citrate or EDTA (© Vacuette). Additionally intravenous catheters were flushed with 0.9 % sodium chloride solution after each sampling time point. Blood samples were drawn before administration and at predefined time points thereafter . Blood samples were then centrifuged at 2000We performed enzyme immunoassays to detect blood levels of hc-DNA. Cell death detection ELISA plus kit (©Roche) uses monoclonal mouse antibodies against histones  and single- and double-stranded DNA  to detect apoptotic or necrotic cells in plasma or cell culture supernatants. In short, histone-complexed mono- and oligonucleosomes are quantified. The presented values are light absorbance values (405 nm) directly derived from the described assay and therefore arbitrary units. The assay was performed according to the manufacturers’ instructions. Histone levels were quantified by Western blotting using antihistone H3 antibodies (Abcam) and calculated using human recombinant histone H3 as standard, as described previously . We perfT tests after logarithmic transformation. Nonparametric tests were not applied because of the small sample size. Where applicable, baseline data were compared with the nonparametric Kruskal–Wallis test, followed by a U test. Correlations were performed by the nonparametric Spearman test. A two-tailed p < 0.05 was considered statistically significant. All statistical calculations were performed using commercially available statistical software (IBM SPSS Statistics version 22).All data are expressed as mean ± standard error of the mean (SEM) unless otherwise stated. For reasons of robustness we have chosen nonparametric tests. Within-group differences were tested overall by Friedman ANOVA, and pairwise comparisons were made by Wilcoxon signed rank test. Results of histone quantification were compared by pairwise We analyzed samples of six participants receiving a placebo, 27 participants treated with 5 µg/kg body weight for four consecutive days and 18 participants receiving a single dose of 300 µg G-CSF intravenously.Baseline characteristics of participants of each study are presented in Table p = 0.003 for overall testing with Friedman ANOVA; compared to baseline, Table A single dose of 300 µg G-CSF significantly increased hc-DNA in the plasma of healthy volunteers 2, 4, 6 and 24 h after the intravenous injection .Subcutaneous injection of 5 µg/kg G-CSF time dependently increased plasma levels of hc-DNA after 24 and 96 h compared to the baseline (P = 0.85) (Table In the placebo group no significant changes in any of the investigated parameters could be detected (5) Table .p = 0.088) 2 h and 1.08 ± 0.30 µg/ml (p = 0.034) 4 h after infusion of 300 µg G-CSF.At the baseline we measured total histone levels of 0.43 ± 0.20 µg/ml, which increased to 1.71 ± 0.54 µg/ml (p = 0.064) was found.In the study investigating effects of four daily doses of G-CSF we detected 1.39 ± 0.37 µg/ml, 24 h after the first G-CSF injection 1.24 ± 0.26 µg/ml and on day five, 24 h after the fourth injection of 5 µg/kg G-CSF, 2.16 ± 0.62 µg/ml. There was no significant difference between these measurements. However, a borderline increase between days two and five (p < 0.001).To examine potential gender differences we performed a pooled analysis of blood samples taken at the baseline. Samples of 26 female and 25 male healthy volunteers were analyzed. We detected significantly higher amounts of hc-DNA in male subjects than in female subjects . In contrast, the trial investigating daily doses of 5 µg/kg G-CSF demonstrated marked gender differences at all times .The sample size of the subgroup receiving a single dose was probably too limited to demonstrate significant gender differences between nine male and nine female volunteers , whereas in male subjects baseline values of 234 ± 67 rose to 319 ± 76 and 838 ± 148 after 24 and 96 h  , and in the study of multiple doses the correlation coefficient was 0.310 (p = 0.005). Figure Although both hc-DNA and neutrophils increased over time, an exploratory correlation analysis including all measured values revealed only a weak correlation within each study. In the single dose study the correlation coefficient was 0.272  that infusion of a single dose of 300 µg G-CSF significantly increases hc-DNA in the plasma of healthy volunteers, (2) that this increase is even more pronounced after injection of four daily doses of G-CSF, (3) that there is a significant difference in serum levels of hc-DNA between male and female subjects, at the baseline and after injection of G-CSF, and (4) histone plasma levels significantly increase as early as 4 h after a single G-CSF injection.Neutrophils of mice receiving daily G-CSF as well as neutrophils of mice bearing G-CSF-producing cancer cells are predisposed to generate NETs after activation . Our finLevels of G-CSF are elevated during infections . UsuallyStaphylococcus aureus is able to produce enzymes, which degrade these NETs and escape this defensive mechanism [A recently discovered defensive mechanism of neutrophil granulocytes called “NETosis” is described as a cell death distinct from apoptosis and necrosis. NETosis is a cell death program that results in the expulsion of decondensated DNA from neutrophil granulocytes alongside the exertion of other preformed substances stored in various granules within the cell. This DNA builds loose congregates of neutrophils, platelets, erythrocytes, and antimicrobial substances as well histones that entangle the pathogens and possibly kill them , 18. Intechanism .In this study we measured hc-DNA before and after G-CSF administration. Although we did not further identify the origin of this circulating extracellular DNA, it seems likely that the DNA is of neutrophil origin. An increase in plasma myeloperoxidase after G-CSF treatment was reported previously. This supports our hypothesis, as this enzyme is not just of neutrophil origin, but can also be found in NETs , 20. TheAbrams et al. pointed out that histones play a pathophysiological role in patients suffering from trauma, but also from necrotizing pancreatitis or sepsis. In their mouse model, infusion of histones led to an increase in thrombin–antithrombin complexes indicating activation of coagulation. Furthermore in 52 trauma patients circulating histone levels were correlated with sequential organ failure assessment (SOFA) scores . In our Beside neutrophils, the activation of the endothelium and endothelial damage may be the source of hc-DNA. G-CSF injection also leads to endothelial activation as demonstrated by Stroncek et al. However, in their study they administered 10 µg/kg G-CSF for 5 days and showed an increase in E-selectin of 78 ± 34 % after 5 days . A similInterestingly, there was a significant difference in the levels of hc-DNA between male and female healthy volunteers, which is in contrast to the results of Tillack et al. . In theiThe role and importance of NETs and circulating extracellular DNA in the pathophysiology of thrombosis are currently intensively investigated –26. A siFurthermore, usually only healthy people before stem cell harvest or patients during neutropenia, which is frequently accompanied by thrombocytopenia, are treated with G-CSF. Thus, thrombotic events are generally rare and more of episodic character. A large trial investigating adverse events in peripheral blood stem cell donors found no increased risk of thrombosis after mobilization with G-CSF compared to bone marrow donors without stem cell mobilization . FinallyInjection of single and multiple doses of G-CSF increased levels of hc-DNA significantly. Interestingly, a gender difference with lower levels of hc-DNA in female than in male subjects was detected.


Toll-like receptors (TLR) are crucial sensors of microbial agents such as bacterial or viral compounds. These receptors constitute key players in the induction of inflammation, e.g. in septic or chronic inflammatory diseases. Colony-stimulating factors (CSFs) such as granulocyte-macrophage-CSF (GM-CSF) or granulocyte-CSF (G-CSF) have been extensively investigated in their capacity to promote myelopoiesis in febrile neutropenia or to overcome immunosuppression in patients suffering from sepsis-associated neutropenia or from monocytic immunoincompetence. We report here that GM-CSF, downregulates TLR1, TLR2 and TLR4 in a time- and dose-dependent fashion in human monocytes. Diminished pathogen recognition receptor expression was accompanied by reduced downstream p38 and extracellular-signal-regulated kinase (ERK) signaling upon lipoteichoic acid (LTA) and lipopolysaccharide (LPS) binding—and accordingly led to impaired proinflammatory cytokine production. Knockdown experiments of the transcription factors PU.1 and VentX showed that GM-CSF driven effects on TLR regulation is entirely PU.1 but not VentX dependent. We further analysed monocyte TLR and CD14 expression upon exposure to the IMID® immunomodulatory drug Pomalidomide (CC-4047), a Thalidomide analogue known to downregulate PU.1. Indeed, Pomalidomide in part reversed the GM-CSF-mediated effects. Our data indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. The PU.1 mediated immunomodulatory properties of GM-CSF should be taken into consideration upon usage of GM-CSF in inflammatory or infection-related conditions. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine with pleiotropic effects. It mainly acts on development and maturation of monocyte/macrophages and granulocytes. Originally this factor was found to promote myelopoiesis , still iIn the clinical entity of sepsis the usage of recombinant CSFs remain controversial. Although rhG-CSF and rhGM-CSF appear to have no adverse side effects, their usefulness in treating and preventing sepsis in both adults and infants remains uncertain as CSF treatment had no impact on mortality . Yet, inA growing body of evidence suggests that GM-CSF also plays an important role in the modulation of immune responses to invading pathogens –15. ForeWe therefore sought to analyse the impact and mode of action of GM-CSF on innate immunity receptors sensing foreign bacteria on primary blood-derived human monocytes. We show that GM-CSF, but not G-CSF, downregulates major bacterial pattern recognition receptors including TLR1, TLR2, TLR4 and CD14 on human monocytes and that this modulation of surface receptors results in impaired downstream TLR signaling—e.g. MAPK phosphorylation. We further investigated the effects of GM-CSF on cytokine production in human monocytes upon challenge with bacterial agents such LPS or LTA.Monocyte/macrophage differentiation is tightly regulated by transcription factors such as c-fos or PU.1 and consistently, PU.1 deficient mice show an arrest in myeloid development . IMiDs® 6/mL) were seeded in Teflon-coated hydrophobic culture plates  and cultured in RPMI 1640 supplemented with 10% FCS and 2 mM glutamine in the presence or absence of indicated GM-CSF concentrations.Heparinized whole blood was drawn from healthy adult volunteers. Human monocytes were separated by density gradient centrifugation and by means of magnetic cell sorting . Monocyte purity of negatively selected cells was > = 95% as determined by flow cytometry. Cells  and proven by the manufacturer to activate exclusively TLR4 as determined with splenocytes and macrophages from TLR4-deficient mice. Staphylococcus aureus-derived LTA (Sigma-Aldrich) was diluted in sterile distilled pyrogen-free water and stored according to manufacturer's suggestions.GM-CSF and G-CSF was purchased from Peprotech . A stock solution of 100μg/ml was prepared in destilled water and stored in small sterile aliquots at -20°C. Effects on cell viability by high concentrations of GM-CSF were assessed by Annexin V-PE/7-AAD  staining. The thalidomide analogue pomalidomide (CC-4047) was kindly provided by Celgene  and a stock solution of 20 mM was dissolved in DMSO. Further dilutions were made in RPMI 1640 shortly before administration to cell culture. LPS purified from 4 CD14+ cells were analyzed by flow cytometry on a Coulter FC 500 equipped with EXPO32 software .PE-labeled anti-human TLR4 , TLR2 , TLR1  as well as corresponding isotype antibodies were purchased from eBioscience . Anti-CD14-mAb  was used to define the monocyte population. Antibody incubation was performed on RT for 30 min. Monocytes were washed twice with Hanks balanced salt solution  containing 0.1% NaN3, 0.3% BSA, and a total of 2×10Intracellular tumor necrosis factor (TNF)-α cytokine staining of CD14+ cells following after 4-h incubation with LPS or LTA was performed with a PE-conjugated anti-human TNF-α mAb . All reagents needed for detection of intracellular cytokines were purchased from BD Biosciences  and intracellular staining was performed according to manufacturer's suggestions. Intracellular TNF-α production  was measured as described above by flow cytometry.Intracellular phospho-specific analysis of SAPK was undertaken as described previously . Briefly6 monocytes were resuspended into 100 μl Nucleofector solution with 0.5 nmol of either PU.1 siRNA  or non-effective GFP siRNA  before electroporation with the Nucleofector II Device (Lonza). Cells were then immediately removed from the device and incubated overnight with 1 ml prewarmed Human Monocyte Nucleofector Medium containing 2 mM glutamine and 10% FBS. Cells were then resuspended in complete RPMI medium and treated with appropriate cytokines to induce differentiation into macrophages.Human primary monocytes were transfected using the Human Monocyte Nucleofector Kit (Lonza) according to the manufacturer’s instructions. Briefly, 5 × 10TM) and 18S rRNA VIC® were obtained predesigned from Applied Biosystems and tested for primer efficacy . Multiplex amplification was carried out in a total volume of 20 μl for 40 cycles of 3 seconds at 95°C, 30 seconds at 60°C. Initial denaturation was performed for 3 min at 95°C. Target gene expression was normalized to 18s rRNA house keeping gene expression. Normalized target gene expression was analysed by the comparative ΔΔ-CT method and calculated as x-fold expression.The ABI PRISM 7500HT Sequence Detection System  was used for qt-RT-PCR analysis. Primer-probes sets for TLR1, TLR2, TLR4 and PU.1  and afterwards incubated with either 10 μg LTA  or 10 ngiability .Multiple transcription factors have been implicated in monocyte activation and therefore might also govern TLR expression. To elaborate the mechanisms underlying GM-CSF-mediated TLR regulation, we employed siRNA technology to knock down the transcription factors PU.1 and VentX expression in primary monocytes. As shown in We next questioned whether pharmacological agents that regulate the transcription factor PU.1 also affect GM-CSF-mediated TLR downregulation. The IMiD Pomalidomide has been shown to down-regulate PU.1 in leukocytes both in vitro and in vivo  and to aToll-like receptors are of major importance in their function as PRRs and thus fulfil the recognition of microbial agents with concomitant immunological stimulation. They have been widely shown to induce an inflammatory response upon pathogen-associated molecular patters (PAMP)- and damage-associated molecular patters (DAMP)-contact and they bridge signalling events between the innate and the adaptive immune response , 21. Howin-vivo on processes from hematopoesis, to angiogenesis, to cellular functionality harbour relevant implications [Since the discovery of the human Toll homologues in the mid 1990s a considerable number of TLR-modifying agents have been described. We previously showed that 1,25-cholecalcitriol—via the Vitamin-D-receptor pathway—is a potent down-regulator of TLR2 and TLR4 in mononuclear cells ; other aications , 26. Allications . In pulmications , 15 and ications . AlongsiThe ever expanding knowledge of the role of TLRs in the pathogenesis of various human diseases make TLRs promising therapeutic targets. The contribution of TLRs in the pathogenesis of atherosclerosis or inflammatory bowel diseases is well documented. , 30 In tPomalidomide (CC-4047) is a thalidomide analogue with immunomodulatory properties. IMiD® compounds pomalidomide and lenalidomide are currently experiencing a renaissance as anti cancer agents for treatment of myelodysplastic syndromes (MDS) and for treatment of patients with multiple myeloma in combination with dexamethasone. These drugs exhibit a range of interesting clinical properties, including anti-angiogenic and anti-proliferative activities although exact cellular targets are only partly understood. Lenalidomide and pomalidomide strongly inhibit T-regulatory cell proliferation and suppressor-function . Also, aIn summary, our findings indicate a critical role of PU.1 in the regulation of TLR1, 2, 4 and of CD14, thus targeting PU.1 ultimately results in TLR modulation. Thus, we identified a novel immunomodulatory function of GM-CSF in human blood derived monocytes. The dampening of proinflammatory TLR signaling pathways might be beneficial in diseases where excessive inflammation leads to harmful outcome. These mechanisms of GM-CSF and TLR interaction might be of relevance for the clinical use of a pleiotropic colony stimulating factor.S1 FigMonocytes were isolated as described in methods and incubated for 48h in the presence or absence of recombinant G-CSF. Cells were harvested and analyzed for CD14, TLR1, TLR2 and TLR4 surface protein by flow cytometry. Error bars show mean fluorescence intensities (MFI) ± SD of three individual experiments.(TIF)Click here for additional data file.S2 FigMonocytes were incubated with or without GM-CSF for 48 hours and Annexin V / 7 AAD staining was performed. Flow cytometry dot plots shown are representative of three individually performed experiments.(TIF)Click here for additional data file.


Calcineurin inhibitors (CNI) have significantly improved patient and graft survival in pediatric liver transplantation (pLT). However, CNI toxicity leads to significant morbidity. Moreover, CNIs cannot prevent long-term allograft injury. Mesenchymal stem  cells (MSC) have potent immunomodulatory properties, which may promote allograft tolerance and ameliorate toxicity of high-dose CNI. The MYSTEP1 trial aims to investigate safety and feasibility of donor-derived MSCs in pLT. 6 MSCs/kg body weight will be given at two time points: first by intraportal infusion intraoperatively and second by intravenous infusion on postoperative day 2. In addition, participants will receive standard immunosuppressive treatment. Our primary objective is to assess the safety of intraportal and intravenous MSC infusion in pLT recipients. Our secondary objective is to evaluate efficacy of MSC treatment as measured by the individual need for immunosuppression and the incidence of biopsy-proven acute rejection. We will perform detailed immune monitoring to investigate immunomodulatory effects.  7 to 10 children undergoing living-donor pLT will be included in this open-label, prospective pilot trial. A dose of 1 × 10 Our study will provide information on the safety of donor-derived MSCs in pediatric living-donor liver transplantation and their effect on immunomodulation and graft survival. In recent decades, pediatric liver transplantation has evolved into a state-of-the-art procedure improving prognosis and quality of life for children and adolescents with terminal liver disease. Immunosuppressive pharmacotherapy including calcineurin inhibitors (CNIs) allows the transplantation of solid organ grafts with reasonable patient and graft survival rates –3. HowevMesenchymal stem cells or, according to the terminology of the International Society of Cellular Therapy (ISCT), multipotent mesenchymal stromal cells , are aduβ1 (transforming growth factor); hepatocyte growth factor [Numerous experimental studies imply that MSCs play a role in modulation of immune responses: in mixed lymphocyte cultures, human MSCs have a significant suppressive effect on T-cell proliferation by means of cell-cell interaction and secretion of soluble factors . Mediatoh factor ; solubleh factor ; and proh factor . Togetheh factor –20, B-ceh factor , and mach factor , which, h factor .Preclinical models have confirmed these beneficial immunomodulatory effects of MSCs , 24, 25.Mesenchymal stem  cells (MSCs) may represent an attractive therapeutic option in solid organ transplantation because they modulate immune response and promote regeneration . SystemiDue to the aforementioned toxicity of CNI and insufficient long-term outcome results after liver transplantation, children may benefit from alternative approaches to immunomodulation that will prolong graft survival while reducing CNI toxicity, improving quality of life and promoting long-term allograft tolerance. However, in the context of pediatric living-donor liver transplantation, the safety and feasibility of intraportal and intravenous application of MSCs remain to be proven.Incidence, timing, and severity of acute complications related to MSC infusion, using a specific toxicity scoring system  and their relation to investigational treatmentGraft integrity and function after liver transplantation, as measured by aminotransferase and gamma glutamyl transferase activity, bilirubin, albumin, and INR.The primary objective of this pilot trial is to assess the safety of donor-derived MSC infusions in children undergoing LDLT. Safety will be determined by the following:Feasibility and safety of tapering immunosuppressive medication according to standard guidelines  and accoTime to first biopsy-proven acute rejection (BPAR).EfficacyHematologic and immunologic function, as measured by characterization and quantification of mononuclear cell populations, detection of donor-specific antibodies (DSA) and liver autoantibodies, and analysis of a protocol transplant biopsy.Patient and graft survival at 1 and 2 years posttransplantation.Further, this study aims to evaluate the following:NCT02957552.The MYSTEP1  trial is a 24-month, nonrandomized, open-label, prospective, single-center pilot trial. In total, a minimum of 7 de novo liver recipients, 0–17 years of age, will be recruited at the University Hospital of Tübingen. We will enroll them in the study upon the consent of their legal representatives and upon meeting the eligibility criteria. The study group may be expanded to as many as 10 patients after consulting the data safety monitoring board (DSMB), which will monitor study progress. The board comprises a pediatrician, a transplant surgeon, and a biometrician not otherwise involved in the trial. We have obtained regulatory approval from the Ethical Committee of the University of Tübingen and from the German Federal Institute for Vaccines and Biomedicines . This trial is registered with Clinicaltrials.gov Patient and both parents and/or legal guardian must have given written informed consent.Patients will undergo living-donor liver transplantation for chronic terminal liver failure.Age ≥ 8 weeks and ≤18 years.Body weight > 5 kg.Patients eligible for inclusion in this study must fulfill all of the following criteria:Living donor not suitable according to donor and recipient criteriaPregnant or breastfeedingRefusal of adequate contraception (if appropriate)Acute liver failure or highly urgent transplantationReceiving any form of solid organ retransplantationMulti-organ-transplantationActive autoimmune disease2 or require hemodialysisPreexisting renal failure with eGFR < 50 ml/min/1.73 mReduced pulmonary function (lung function test in children older than 6 years: FEV1 and FVC < 70% of age-appropriate norm) or clinical suspicion of pulmonary disease affecting the patient's physical performance, requiring invasive or noninvasive mechanical ventilationHistory of pulmonary embolismPulmonary hypertension and/or right ventricular load in echocardiographyReduced cardiac function: left ventricular shortening fraction < 25%Clinically significant systemic infectionsUndergoing critical care treatment like mechanical ventilation, dialysis, or vasopressor agentsSeropositivity for HIV, HTLV, or hepatitis B/CHepatobiliary malignancies or history of any extrahepatic malignancyThrombophiliaBudd-Chiari syndromePreexisting thrombosis of portal veinDoppler-sonographic evidence for relevant portosystemic shunts, for example, persistent ductus venosusCold ischemia time > 90 minKnown abuse of drugs or alcoholKnown allergy to DMSO.Patients fulfilling any of the following criteria are not eligible in this study:6 cells with 20 ml bone marrow. Only in rare occasions, more bone marrow is needed for MSC therapy in pediatric recipients.Donor-derived mesenchymal stem cells will be obtained 4 weeks before the planned LDLT via bone marrow puncture of the living donor. All donors will undergo routine examination and screening tests including an extensive infectious disease work-up, according to our transplant center's donor screening protocol , 38. Don6 cells/ml.The cell product can only be released if the following criteria have been fulfilled: regular surface marker expression ; spindle-shaped morphology; a colorless cell suspension; viability of cells of >80%; absence of microbial contamination using culture, mycoplasma PCR, and endotoxin testing; and absence of cell aggregates. The MSC product is cryopreserved with 10% DMSO until designated application (storage at <−150°C in gas phase of liquid nitrogen). On the day of cell transfusion, the assigned dose of mesenchymal stem cells will be thawed at the GMP stem cell laboratory, washed, and suspended in an appropriate volume of isotonic saline with 0.5% albumin at a cell concentration ranging between about 1 and 1.5 × 106 MSC/kg body weight, with the first infusion intraoperatively (day 0) and the second infusion postoperatively, on days 1–3 after living-donor liver transplantation  during the screening visit.Children enrolled in this study will undergo a standard pretransplant work-up, which consists of baseline clinical data . Pregnancy tests will be performed using a test for 6 cells/kg body weight will be performed intraoperatively via portal infusion after complete liver allograft reperfusion (day 0). MSC suspension will be administered via a small venous catheter into the portal vein for 20 minutes while gently waving the syringe to keep cells in suspension. In addition to using Doppler ultrasonography, we will measure portal flow by transit time flow measurement (Medistim®) during cell infusion [6 cells/kg body weight, will be performed via a systemic intravenous route on postoperative day 2 (time window ± 1 day). MSC suspension will be transfused via a central venous catheter or peripheral venous catheter for 20 minutes under sterile conditions. During cell infusions, we will continuously monitor the patient's pulse, blood pressure, oxygen saturation, respiratory rate, and body temperature.The first administration of 1 × 10infusion , 40. CelWe will see patients frequently for follow-up visits during the first 28 days after transplantation. Study visits will consist of regular clinical examinations, Doppler sonography, and blood tests aimed at early detection of treatment-emergent events. We will assess the patient's MYSTEP toxicity score on days 0, 2, 4, 7, 10, and 28 after LDLT. Additional study visits will be performed up to 720 days after LDLT to assess allograft survival, incidence of rejection, incidence of (opportunistic) infections, kidney function, and individual need for immunosuppressive medication . If the In order to evaluate and quantifiy treatment-emergent adverse events of MSC infusion, we defined a pediatric infusional toxicity score that adopts the MiSOT-I score for adults . The scoIn this study, we will perform a protocol liver biopsy 6 months after LDLT and MSC infusion. A percutaneous liver biopsy will be performed under sonographic control and in analgosedation, according to recommendations of the ESPGHAN Hepatology Committee  and locaAdditional blood samples will be collected to investigate surrogate markers of the participant's immune response status . This imMSCs may support induction of allograft tolerance and help to achieve long-term tolerance . PatientIntroduction of current standard immunosuppressive therapies including CNIs has had a major impact on reduction of acute mortality after pediatric liver transplantation , 50. HowTo our knowledge, this is the first clinical trial of immunomodulating therapy with mesenchymal stem cells in pediatric solid organ transplantation. In this pilot study, we aim primarily to determine safety and feasibility of intraportal and intravenous infusion of donor-derived MSC in children undergoing LDLT. We will assess safety based on incidence of acute infusion-related complications measured by the MYSTEP score, on occurrence of severe adverse events and on allograft function after pLT. We have designed our study protocol to optimize prevention of adverse events and to identify any that arise early. For example, using a “staggered approach” to patient recruitment and ensuring intraoperative quantitative monitoring of graft perfusion by transit time flow measurement will help ensure a very high level of safety for the children who participate. In adults, no toxicity has been observed during intravenous and intraportal infusion to date .Previous studies of MSCs in kidney transplantation have shown contradictory data on the risk of overimmunosuppression, which may lead to opportunistic infections , 31. ThiMany details of the mechanisms of immunoregulation by MSCs in transplant recipients remain unknown. It is evident, however, that an increase in the percentage of Foxp3 positive regulatory T-cells is one important mode of MSC action in transplant patients , 35, 52.The optimal donor source of MSCs is still unclear. In this trial, we will use bone marrow-derived MSCs obtained from the solid-organ donor. Donor-derived MSCs may contribute to donor-derived allograft tolerance . Living-In addition to the source of MSCs, the time and route of cell application may influence the effectiveness of MSCs. Direct infusion of the cells into the graft can make use of the tissue-repair capacity of MSCs to treat ischemia reperfusion injury. Furthermore, preclinical models showed that in addition to systemic effects, local mechanisms were responsible for transplant tolerance by MSCs . One impIn keeping with our focus on safety and feasibility, all participants will be treated in combination with the center's standard immunosuppressive regime, which consists of basiliximab, tacrolimus, and steroids. Preclinical studies suggested that CNIs and glucocorticoids may affect MSC morphology, migration, and immunomodulatory behavior, possibly affecting the success of the cell therapy , 60. HowIn conclusion, MSCs have the potential to become part of an array of novel treatment options for pediatric LDLT recipients aimed at promoting allograft tolerance and improving long-term allograft survival while reducing toxicity of chronic IS treatment. A positive outcome of the MYSTEP1 trial in terms of safety and allograft survival would constitute a major advancement in pediatric solid organ transplantation. Subsequently, we intend to conduct a second, larger multicenter trial to study the immunomodulatory efficacy of MSC treatment protocols for improving long-term allograft tolerance in pediatric liver transplant recipients.


In mucopolysaccharidoses (MPS), glycosaminoglycans (GAG) accumulate in tissues. In MPS II, approximately two-thirds of patients are cognitively impaired. We investigated levels of GAG in cerebrospinal fluid (CSF) in different populations from four clinical studies (including NCT00920647 and NCT01449240). Data indicate that MPS II patients with cognitive impairment have elevated levels of CSF GAG, whereas those with the attenuated phenotype typically have levels falling between those of the cognitively affected patients and healthy controls. Patients were aged < 18 years, with cognitive impairment due to MPS II . CSF samples were obtained at screening  2.A multicenter study of pediatric and adult patients with MPS II . All pediatric patients were scheduled to undergo a non-study-related lumbar puncture or other medical or diagnostic procedure that required the administration of general anesthesia during which a lumbar puncture could be performed 3.A single-center study of normal healthy adult volunteers undergoing lumbar puncture (HGT-HIT-073). Patients were ≥ 18 and ≤ 30 years of age, and with no clinically significant findings.4.Samples from surrogate-normal children  were obtained from two sources. One was a multicenter, noninterventional study of male or female pediatric subjects (< 18 years of age) undergoing a procedure allowing access to CSF for other clinical reasons (HGT-HIT-083). Age groups included newborns (0–27 days), infants and toddlers (28 days to 23 months), children (2–11 years), and adolescents (12 to < 18 years). The second source was 157 samples from surrogate-normal children obtained from a biorepository at The Children's National Medical Center (CNMC) in Washington DC, and these were analyzed with the HGT-HIT-083 samples.The data presented here are from four studies:Informed consent was obtained from all patients and/or legal guardians and independent review board/independent ethics committees approved all studies. Study procedures complied with Good Clinical Practice as described in the 21 CFR Parts 50, 56, and 312 and the International Conference on Harmonisation Guidelines, as well as with the ethical principles described in the Declaration of Helsinki.2.1CSF samples were evaluated for glucose, protein, albumin, and cell counts. CSF samples were stored frozen for subsequent analysis of total GAGs. Total CSF GAG concentration was determined by Shire  using a thrombin activity assay with chromogenic substrate S-2238™ . CSF samples were pre-incubated with a fixed amount of human heparin cofactor II , then incubated with a fixed amount of Human α-Thrombin  and 0.5 mM S-2238 in assay buffer. GAG in CSF samples binds to heparin cofactor II, which in turn accelerates thrombin inactivation. The GAG concentration was calculated from a calibration curve prepared from dermatan sulfate  included in the same assay. The lower limit of quantification was 36.7 ng/mL. The assay mainly detected dermatan sulfate and heparan sulfate in the GAG family. This assay was used to measure the CSF GAG levels for all studies reported in this paper. The quantification of urinary GAG for HIT-045, HIT-072 and HIT-073 was performed by Cambridge Biomedical Inc.  using a Blyscan™ Glycosaminoglycan Assay kit , and results were normalized to urine creatinine 2.2Descriptive summaries were presented for the following subject groups: surrogate-normal children, healthy adults, cognitively-intact MPS II patients, and cognitively-impaired MPSII patients. Data for surrogate-normal children from study HGT-HIT-083 were merged with those from the biorepository samples and analyzed in 4 age groups.3Two hundred and fifty seven subjects from the 4 studies were assessed, and their baseline or Day 1 characteristics were measured. Data from study HGT-HIT-072, which enrolled both cognitively intact and cognitively impaired children with MPS II, as well as cognitively intact adults with MPS II are not shown separately, but per cognitive category. For instance, the data from the 3 cognitively impaired children whose CSF was obtained in study HGT-HIT-072 . In the HGT-HIT-045 study all patients had received and tolerated a minimum of 6 months of treatment with intravenous idursulfase, prior to starting the study, so urinary GAG levels would not be expected to reflect accurately any baseline levels. In the HGT-HIT-083 study, no other GAG measurements were taken, as the study activities were limited to obtaining samples of CSF. None of studies measured serum GAG levels. For these reasons there is insufficient data to perform correlation analyses of urinary GAG and CSF GAG levels in these studies.4Dekaban and his colleagues initially reported in 1973 that subjects with MPS and unimpaired intellects had lower levels of CSF GAGs than MPS subjects who were mentally impaired Dr. Christian Hendriksz has received consulting fees from Actelion, BioMarin, Glaxo-SmithKline, and Sanofi-Genzyme, and has undertaken contracted research for Actelion, Amicus, BioMarin, GlaxoSmithKline, Sanofi-Genzyme, Shire, and Synageva. Dr. Joseph Muenzer has been a consultant to BioMarin, Shire, and Zacharon, and serves on advisory boards and speakers bureaus for Genzyme, BioMarin, and Shire. He is also currently the principal investigator for phase 1/2 and phase 2/3 IT ERT clinical trials for MPS II sponsored by Shire. Dr. Adeline Vanderver has received consulting fees from Shire and has performed (unpaid) consulting for Stemcells, Inc. Dr. Jonathan Davis has no conflicts to declare. Dr. Barbara Burton has received consulting fees from, and serves on advisory boards for, BioMarin and Shire, and has undertaken contracted research for BioMarin, Cytonet, Genzyme, Shire, Synageva, and Ultragenyx. Dr. Nancy Mendelsohn has received grant funding from Genzyme, BioMarin, and Shire. Ms. Nan Wang and Dr. Luying Pan are employees of, and own stock in Shire. At the time of these studies Drs. Arian Pano and Ann Barbier were employees of Shire and they own stock in Shire.


Digital Polymerase Chain Reaction (dPCR) is a novel method for the absolute quantification of target nucleic acids. Quantification by dPCR hinges on the fact that the random distribution of molecules in many partitions follows a Poisson distribution. Each partition acts as an individual PCR microreactor and partitions containing amplified target sequences are detected by fluorescence. The proportion of PCR-positive partitions suffices to determine the concentration of the target sequence without a need for calibration. Advances in microfluidics enabled the current revolution of digital quantification by providing efficient partitioning methods. In this review, we compare the fundamental concepts behind the quantification of nucleic acids by dPCR and quantitative real-time PCR (qPCR). We detail the underlying statistics of dPCR and explain how it defines its precision and performance metrics. We review the different microfluidic digital PCR formats, present their underlying physical principles, and analyze the technological evolution of dPCR platforms. We present the novel multiplexing strategies enabled by dPCR and examine how isothermal amplification could be an alternative to PCR in digital assays. Finally, we determine whether the theoretical advantages of dPCR over qPCR hold true by perusing studies that directly compare assays implemented with both methods. In this section, we present the basic concepts that underlie the quantification of nucleic acids by digital and quantitative real-time PCR. An experimental comparison between the two methods will be detailed in n copies can, in theory, be produced after n cycles. In practice, the amplification process saturates and reaches a plateau as PCR reagents are depleted and accumulated PCR products self-anneal, preventing any further amplification. Conventional PCR analyzes amplified products at the end of the reaction using gel electrophoresis (end-point measurement).Polymerase Chain Reaction (PCR) is an in vitro technique that amplifies DNA, generating several millions of copies of a specific segment of DNA from a minute amount of starting material . Its speReal-time PCR is based on PCR and measures the amount of PCR product after each round of amplification using a fluorescent readout . A typicWhile qPCR may be labor-intensive and suffers from limited reproducibility ,5,6,7; iDigital polymerase chain reaction (dPCR) enables the absolute quantification of target nucleic acids present in a sample and alleviates the shortcomings of qPCR ,9,10. InThe key difference between dPCR and qPCR lies in their strategy to measure the amount of target sequence. In qPCR, the reaction is monitored throughout the amplification process, and quantification is based on the analysis of the fluorescent signal at the exponential phase. In contrast, dPCR collects fluorescence signals via end-point measurement and uses the number of positive partitions over the total to back-calculate the target concentration . dPCR reIn brief, dPCR is a method of absolute nucleic acid quantification that hinges on the detection of end-point fluorescent signals and the enumeration of binomial events (absence (0) or presence (1) of fluorescence in a partition)  13]. Th. Th13]. e.g., absolute quantification or copy number variant analysis.dPCR benefits from statistical foundations that permit to infer both the target concentration and the accuracy of the quantification. This section reviews the statistical approaches underlying quantification by dPCR. Those approaches depend directly on the specific applications of the dPCR assay, p of a partition to contain at least one target sequence, we should consider the case of the random distribution of m molecules into n partitions. This situation corresponds to a binomial process where the outcome of each drawing is either present or absent and the drawing is repeated m times. The chance of a target sequence to be present in a partition is p is the complementary chance of the partition to be empty after the m target sequences are distributed. A partition has m chances, or attempts, to receive one target sequence. The chance for a partition to be empty is then m attempts, finally p can be approximated to k (containing some target sequences) over the total number of partitions n is sufficient to predict the initial concentration of the target sequence in the sample with To estimate the probability Intuitively, the confidence in the estimation of the target concentration depends on the number of empty partitions. In extreme cases, i.e., when most of the partitions are either empty or full, the confidence in the estimated concentration is very low because the pattern empty/full is not very informative.p is the probability that a partition is empty, n the total number of partitions and The confidence interval is typically estimated using functions that can be directly calculated. Those estimations rely on assumptions that have direct consequences on the estimation. For instance, the Wald method approximates the binomial distribution (a discrete function with finite support) with a normal distribution (a continuous function with infinite support) ,15. As ay solved . Furthery solved .The previous considerations suggest that there exists a value of lambda for which the initial template concentration can be estimated with the highest confidence. In cases of 10,000 or more partitions, the maximal confidence is obtained for a λ value of about 1.6, which corresponds to a proportion of 20% of empty partitions . As notertitions , insert.For over a century, digital assays were conducted to estimate the concentration of microorganisms of public health concern ,19,20,21dPCR has been extensively used to measure genetic imbalances, or Copy Number Variant (CNV), that result from the deletion or amplification of genomic regions or locus. In CNV analysis, the copy number of a locus relative to another is the relevant information. The statistics to estimate the presence of a genetic imbalance using dPCR employs various methods. One such method relies on the Sequential Probability Ratio Test (SPRT), initially developed for quality control. It continuously tests two concurrent hypothesis while accumulating data until a hypothesis is considerably more probable than the other . In dPCRThe analysis is only performed on a sample, which is a small portion of a specimen. This imposes a fundamental limit on the quantification accuracy. The concentration of an analyte in the tested sub-volume may differ greatly from the concentration of the analyte in the entire specimen due to statistical sampling ,35. In oPoisson statistics relies on two assumptions: (1) target sequences are randomly distributed across partitions and (2) all partitions have the same volume. The random distribution of target sequences has been validated experimentally by deriving the Ripley’s K function that measures the randomness of the spatial distribution of partition occupancy ,37, or bQuantification by dPCR assumes that partitions possess identical volumes; however, a large degree of variability in volume may be observed. The latter directly depends on the methods used to create the partitions. The effect of this variation in partition volume has been experimentally assessed and considered a potential source of dPCR imprecision . For λ hdPCR is a statistical method that divides a sample into numerous partitions and enables the enumeration of empty and occupied partitions to determine the concentration of a target sequence present in a sample. dPCR is an absolute quantification method that does not rely on calibration curves and whose accuracy is more easily predictable . The theThe sensitivity or lower limit of detection corresponds to the detection of a single molecule in a single partition. Hence, the minimal concentration that can be detected depends on the total volume of the reaction or on the number of partitions and their volume. This simple reasoning underlines the limits of sensitivity of dPCR because dPCR techniques rely on partitions with volumes in the pL-nL range and their number is limited in practice. By contrast, the reaction volume of qPCR is typically much larger and can also be easily adjusted to reach higher sensitivity.6 partitions created . I. I69]. IThe actuation can also play a key role in simplifying an experimental set-up. For instance, spinning can distribute fluid into chambers located along a spiraling channel . UnfortuIn the self-digitization approach ,74, the The process contrasts with passive partitioning because it involves the formation of droplets that are generated through the splitting of a plug through a network of chambers , and it The first goal of emulsification is to create isolated microreactors of aqueous droplets within immiscible oil. A critical component of this technology is the surfactant and oil formulation that assures both the stability of those microreactors and their compatibility with molecular reactions such as PCR or isothermal amplification .Encapsulation does not always perform sample partitioning and it can be used just to create independent microreactors such as in BEAMing  ,84,85. IMicrofluidic droplet methods differ from BEAMing by using droplets as true partitions. They are enabled by microfluidic emulsification techniques that generate monodisperse droplets with very limited volume variation. Microfluidic droplets can be created with different techniques such as T-junction , nozzle dPCR applications based on microfluidic droplets have been enabled by single molecule amplification ,95,96,97The throughput of droplet digital PCR (ddPCR) is often limited by the readout that is typically performed by interrogating droplets sequentially in a configuration inherited from flow cytometry b. The reMinimizing the need for specialized equipment to perform partitioning represents an important technological trend. Microfluidic droplets have been generated using gradient of confinement, a method similar to step emulsification that permits to simplify actuation of the oil phase d 87]. I. I87]. IA wide range of microfluidic approaches has been used to implement dPCR . dPCR plCost-effective manufacturing is also a critical factor for high volume or commercial applications, and plastic injection molding is preferred over PDMS. There is a trend towards simplifying the actuation, which will lower the cost of those platforms, reduce the hands-on time and support the widespread adoption of dPCR. dPCR platforms currently lack the sample multiplexing capability of qPCR.Similarly to qPCR, dPCR uses two main types of chemistries for the detection of nucleic acid: DNA intercalating dyes and hydrolysis-based probes . Both deMultiplexed qPCR assays permit to simultaneously detect multiple targets in a single reaction using probes labeled with different fluorescent dyes. The number of target sequences that can be quantified concurrently by qPCR is thus spectrally limited by the spectral bandwidth of detection and the fluorescent characteristics of the dyes.In multiplexed dPCR assays, the different target sequences can be coded not only with different fluorescent colors but also with different fluorescence intensities ,105. ThiThe sample can be encapsulated directly with the different assays in a one-step manipulation because dPCR avoids template competition. Multiplexed dPCR assays highlight the importance of robust algorithms for data thresholding and clustering . MultiplAlthough PCR is established as the method of choice for molecular diagnostics, it presents several fundamental limitations such as high cost of equipment and sensitivity to inhibitors . These lA wide variety of isothermal nucleic acid amplification technologies have been developed such as rolling circle amplification (RCA) , nucleicvia ligation of padlock probes, the target templates are amplified using RCA and labeled with fluorophore molecule-tagged probes. The system was successfully applied for the quantification of Vibrio cholera, the causative agent of cholera. A digital RPA has been implemented on a SlipChip device allowing more than 1000 nanoliter-scale RPA reactions to occur simultaneously. The performance of the system was validated by detecting a single molecule of the methicillin-resistant Staphylococcus aureus (MRSA) genomic DNA [E. coli from urine specimens [A digital quantification system using RCA enables the detection of nucleic acids or proteins at the single-molecule level . After comic DNA . SlipChiomic DNA ,123. Recpecimens . dLAMP wpecimens . A digitpecimens . EXPAR, pecimens .Some of these studies have compared the performance of digital isothermal amplification with dPCR. Digital MDA was found to be several orders of magnitude more sensitive than dPCR to quantify contaminant DNA. This result emphasizes the fact that MDA is not sequence-specific and unlike PCR, it does not require intact genomic DNA to generate a positive signal . dLAMP wE. coli DNA using a centrifuge-driven emulsification approach [E. coli in urine samples within 7 min, while it required 2 h with droplet dPCR [In other instances, dLAMP and dPCR provided very similar results for the detection of approach . A dLAMPlet dPCR . Despitelet dPCR . Howeverlet dPCR . dPCR maOverall, few studies have thoroughly compared digital assays based on isothermal amplification and PCR. This stems from the fact that isothermal NAATs and digital assays are both very recent technologies. There is a need for detailed evaluation and comparison of isothermal amplification with PCR in digital assays. Thus far, digital isothermal quantification does not necessarily demonstrate superior performance when compared to dPCR and have not fully delivered on their promise of simplified assays. Digital isothermal quantification relies on multi-step workflows, may require assays to be prepared at 4 °C to prevent spurious amplification or necessitate tight temperature control for optimal amplification . DigitaldPCR is expected to show higher resilience to inhibitors because target sequences are efficiently concentrated in smaller volumes. Several studies have reported the higher tolerance of dPCR to diverse types of inhibitors as compared to qPCR ,130,131.dPCR outperforms qPCR in the analysis of copy number variation ,33,105, BCR-ABL when combined with a pre-amplification step [A comparative evaluation of the performance between an established real-time qPCR assay and a droplet dPCR assay showed comparable detection sensitivity for the quantification of HIV-1 DNA with either droplet dPCR  or the Sion step , but didion step . The lowion step ,125,127.ion step . These rThe calibration-free nature of dPCR should confer an advantage on assay reproducibility . In compDespite the relatively limited number of thorough studies that compare the performance of dPCR and qPCR side-by-side, a few key points emerge: (1) small partition volume contributes to dPCR resilience to a large variety of inhibitors; (2) dPCR is more precise for quantifying relative abundance ; (3) dPCR suffers from lower sensitivity for absolute quantification, which is attributed to its smaller reaction volume. A key argument that could support the widespread use of dPCR in clinical settings would be its expected high technical reproducibility; however, day-to-day and inter-laboratory reproducibility of dPCR has yet to be rigorously assessed.dPCR reduces the quantification of a target sequence to the enumeration of a series of positive and negative amplification reactions, thus converting a continuous or analog signal into a series of binary or digital signals. dPCR has been enabled by advances in microfluidics that provide efficient methods to create many independent reactors.It is critical to understand the statistical foundations of dPCR to interpret data and appreciate the design parameters that define its performance. The key design parameters of dPCR platforms include the number of partitions, the volume of individual partitions, the total reaction volume and the variation of partition volume. The statistical precision of dPCR is further compounded by the variability of sample preparation and the rate of molecular dropouts and false positives. A wide range of microfluidic approaches has been used to develop dPCR platforms. The partitioning methods inform on the source of volume variation, the density of partitions that can be created and the simplicity of the experimental set-up. Through the rapid evolution of the platform technologies there is a clear trend towards simpler actuation, higher density of partitions, larger reaction volume, and the use of microfabrication methods that support high volume manufacturing.dPCR platforms currently lack the sample multiplexing of qPCR while providing unique assay multiplexing capabilities. Isothermal amplification is an attractive alternative to PCR to reduce the complexity of the instrument and to further improve tolerance to inhibitors. Thus far, none of the emerging isothermal amplification techniques has exceeded PCR in terms of performance and assay workflow. Finally, even though only a limited number of studies directly compare the performance of dPCR and qPCR, it appears that dPCR is more resilient to inhibitors and provides higher precision for quantifying relative abundance  of target sequences. However, dPCR currently exhibits lower sensitivity compared to qPCR. dPCR is an emerging technology that may outperform qPCR in clinical applications thanks to its robustness and promised technical reproducibility. Microfluidic technologies have played a central role in enabling the revolution of digital quantification. They provided efficient methods to perform sample partitioning, which is at the core of the dPCR concept. Importantly, microfluidics is a very active field that keeps providing new and creative solutions to improve current platform performance.