After measurement for those analytes, our data suggested that IgA-based ELISA could be adapted for quantitative measurement of those analytes existing as major serum proteins or as multi-protein complexes like RBP4.Elevated circulating Retinol-binding protein 4 (RBP4) has been associated with insulin resistance, dyslipidemia, and hypertension. However, many commonly used RBP4 ELISAs have limited dynamic range. We therefore developed an enzyme-linked immunosorbent sandwich assay (ELISA) employing a novel immunoglobulin A (IgA)-type capture mAb called AG102 instead of IgG subtypes, which was selected for its stability, capture efficiency, and specificity for human RBP 4. These features of RBP4 have hampered the development of quantitative immunological assays. Molecular analysis of AG102 revealed IgA heavy and light chains and a J chain, as expected. AG102 demonstrated notable detection of both bacterial- and HEK293-expressed RBP4 in Western blots. Serial and internal deletion experiments suggested that a putative epitope may be located in the first 35 amino acids of the mature RBP4. Compared with commercial ELISAs, the AG102-based system exhibited more significant recovery of RBP4 from serum or urine at any given dilution factor. To substantiate its quantitation capacity, comparison between RBP4 measurements from quantitative western blots and the AG102-based ELISA demonstrated a significant correlation (R Serum retinol binding protein (RBP4) is increased in insulin-resistant states and highly associated with both the magnitude of insulin resistance and individual components of the metabolic syndrome and the risk to develop coronary heart disease in humans5. Increased serum RBP4 causes insulin resistance in mice by interfering with insulin signaling in skeletal muscle and the liver6. In addition, RBP4 activates antigen-presenting cells in adipose tissue, thereby eliciting low-grade inflammation and leading to systemic insulin resistance7. This suggests that RBP4 is an important regulator for immunometabolism. RBP4 is expressed in the liver and adipose tissue, and adipocyte RBP4 mRNA is strongly increased in human obesity, particularly within visceral fat deposits5. Recent studies indicate that a functional polymorphism of the RBP4 promoter causes increased adipocyte RBP4 expression and is associated with increased serum RBP4 concentrations and risk for type 2 diabetes10. Noteworthy, some studies did not find a relationship between circulating RBP4 level and insulin resistance13. These discrepant findings may reflect differences in study subjects, differences in methods used for quantifying insulin resistance, or technical problems inherent in the current methods used for measuring RBP414.Insulin resistance contributes to the progression from normal glucose tolerance (NGT) to impaired fasting glucose (IFG), impaired glucose tolerance (IGT), and type 2 diabetes mellitus (T2D)14. Several factors may influence the ability of different assays to accurately quantify RBP4. In healthy individuals, the majority of circulating RBP4 exists in a stable 1:1 complex with Transthyretin (TTR), also known as prealbumin, a 56 kDa plasma protein15. RBP4 binds TTR with high affinity via sites on both proteins that have been identified by X-ray crystallography of the complex15. RBP4 ·TTR binding stabilizes RBP4 in circulation by preventing its glomerular filtration16. The carboxyl terminus of RBP4 forms part of the TTR binding interface, and carboxyl terminus-proteolyzed RBP4 variants display reduced affinity for TTR15. Proteolyzed RBP4 appears to undergo rapid renal clearance, since it is abundant in urine, but difficult to detect in normal individuals. Conversely, proteolyzed RBP4 variants have been found to accumulate in the serum of individuals with end-stage renal disease who lack an effective clearance mechanism18. It is likely that the relative concentrations of RBP4 and TTR in circulation, the affinity of RBP4·TTR binding, and the variable presence of proteolyzed RBP4 can influence the ability of immunoassays to accurately quantify RBP4. In addition to this diagnostic pitfall, RBP4 serum concentration is typically higher than a few hundred µg/ml, resulting in the so-called “hook effect” even after significant dilution19.Accordingly, quantitative Western (q-western) blotting has remained a preferred method for RBP4 serum analyses15. The subsequent extensive validation assays exhibited greater dynamic recovery of RBP4 from serum and urine than other commercial assays. The distribution features of serum recovery via this IgA-based ELISA were comparable to those of q-Western blotting. We propose that this novel IgA-based immunoassay should be used to define the relationships between urinary RBP4 level and clinical measures of insulin-glucose homeostasis and urinary albumin excretion.Here, we report the development of a new enzyme-linked immunosorbent sandwich assay (ELISA) using a novel IgA mAb for efficient capture of human RBP4 from serum or urine. Due to its dimeric structure, IgA’s antigen-capture ability is superior to that of IgG. The current IgA mAb was selected for its reactivity with a specific epitope of RBP4 at the N-terminus, which is not located within the RBP4·TTR binding interface- terminus of the peptide. The augmented cDNA of RBP4 was then cloned into pAGNF and pET-21a(+) vectors  and Nde I/Xho I were used for constructing the expression vector. pAGNF is driven by the CMV early promoter, and protein secretion is facilitated by the plasminogen activator inhibitor type I (PAI-1) leader peptide20. FLAG-RBP4 was expressed in a human embryonic kidney cell line, HEK-293, and purified from conditioned media using an anti-FLAG column . 6 × His-tagged RBP4 was purified via a Ni Sepharose column. A polyclonal antibody (pAb) and mAbs were produced by immunizing rabbits and BALB/c mice, respectively, with recombinant 6X His-tagged human RBP4, and hybridomas were generated and cloned according to standard protocols21. A panel of mAbs produced by different clones was screened with respect to reactivity to FLAG-RBP4. For epitope mapping, two deletion versions of recombinant human RBP4 lacking amino acid residues 1–35 or 138–183 and an internal deletion mutant lacking amino acid residues 36–78 were created by PCR with appropriate primer sets  was used according to the manufacturer’s instructions.The cDNAs for the variable genes of the heavy and light chains of AG102  were amplified using the 5’ rapid amplification of cDNA ends (5’RACE) method. Primer sequences are presented in Supplementary Table A sandwich ELISA format was designed using AG102 as the capture antibody and pAb  for detection, with FLAG-tagged RBP4 as a standard. The wells of a microtiter plate were first coated with 5 μg/ml AG102 at 37 °C for 1 h. Next, 100 μl of human serum , human urine (diluted 1:100), or diluted standard was applied to the AG102-coated wells and incubated at 37 °C for 1 h. The wells were then washed three times with 0.05% Tween-20 in phosphate-buffered saline (PBST). Next, 100 µl of the purified pAb (5 μg/ml) was added per well and incubated at 37 °C for 1 h, and the wells were washed three times with PBST. Detection was performed by incubating each well with 100 µl of horseradish peroxidase (HRP)-conjugated streptavidin (Zymed) diluted 1:1,000 in PBS at 37 °C for 1 h, washing three times in PBS, and conducting a colorimetric reaction for 20 min with the HRP substrate 2,2′-azino-bis(2-ethylbenzothiazoline-6-sulfonic acid) . Optical density was measured at the product’s maximal absorbance of 450 nm. All participants provided written informed consent. The Institutional Review Board of Kangbuk Samsung Hospital in Korea approved the study protocol that was in accordance with the Declaration of Helsinki.2 according to the manufacturer’s instructions.Predetermined recombinant RBP4 proteins expressed by HEK293 cells were resolved on a polyacrylamide gel in parallel with five serum samples whose concentrations were determined by the IgA-based ELISA. After probing the Western blot filters with a rabbit anti-RBP4, phospho-image analysis was conducted using an image analyzer . Quantitation is represented in AU/mm15. The cDNA sequence corresponding to the J chain was also verified. These findings confirm that AG102 is a monoclonal IgA that recognizes an N-terminus epitope of RBP4 that is likely to be exposed on the surface of RBP4 and unlikely to be affected by either high-affinity RBP4·TTR interactions or RBP4 C-terminal proteolysis, factors proposed to contribute to variability and technical limitations in other assays15.As shown in Fig. 2 = 0.859). To compare the correlation and dynamic serum recovery associated with the IgA-based ELISA, 20 serum samples were subjected to quantification with the IgA ELISA and with commercial ELISAs. As seen in Fig. 2 = 0.883, P < 0.001), indicating that the novel assay was at least comparable in performance to commercial assays with regard to measurement of serum RBP4 concentration14. However, the dynamic range of serum RBP4 concentrations detectable by the IgA-based ELISA was significantly larger than that of the commercially-available ELISAs. We routinely observed that the degree of serum recovery via the IgA-based ELISA was 1.5- to 2.0-fold larger than that of the commercial assays.A classic sandwich ELISA using AG102 as the capture antibody was created and used to measure RBP4 in five selected human sera exhibiting a broad range of RBP4 concentrations. The maximum sensitivity was 380 pg/ml. Intra-assay C.V. was 1.7–3.7% . In addition to this unique feature, intra- and inter-assay coefficients of variation were below 4% and 8%, respectively, suggesting that this IgA-based ELISA was highly stable  ELISA and two commercial assays . Measurements of urinary RBP4 with the new ELISA correlated significantly with the commercial assay 16. In addition, other studies15,23 have demonstrated that the RBP4 C-terminus, which plays an important role in forming the RBP4·TTR interface, is proteolyzed to varying degrees in serum and urine. In addition to influencing the RBP4·TTR interaction, proteolysis of RBP4 may influence RBP4 conformation. Together, these factors could affect the accessibility of any given monoclonal antibody or affinity-purified polyclonal antibody to native RBP4 obtained from serum or urine, especially when studied under diluted conditions, resulting in variable recoveries of RBP4.Here, we demonstrated a newly developed IgA mAb-based ELISA, which is a semi-quantitative tool for measuring the major serum protein, RBP4, upon a proper single dilution. To our knowledge, this new immunologic assay is the most convenient option that has been described to replace q-Western blot or LC/MS-MSOur analysis clearly shows that the IgA mAb AG102, which is directed toward the extreme N-terminus of RBP4 far from the regions of RBP4 known to be involved in TTR binding, displays a superior recovery of urinary RBP4 compared to commercial RBP4 assays utilizing IgG antibodies. In addition, it is likely that the greater tolerance of IgA for a wide range of pH values and the greater number of antigen binding sites on IgA may provide a superior capture antibody for measuring urinary RBP4. Thus, this newly developed IgA-based ELISA will provide a more reliable method for measuring urinary RBP4 and thereby enhance our understanding of the relationship between RBP4 and impaired insulin-glucose homeostasis. Since our cDNA sequences revealed the CDR and framework regions, these regions can be replaced by a specific antigenic-binding site and engineered to generate synthetic IgA-based mAbs, thereby helping improve this IgA technology.Supplementary information


Among patients presenting with diverse neurological problems at a single center, cerebrospinal fluid discordance or escape was observed in 15% and was associated with diffuse white matter signal abnormalities on cranial magnetic resonance imaging. 10 copies HIV-1 RNA greater than plasma VL) or escape (detection of HIV VL >50 copies/mL in CSF in patients with suppressed plasma VL <50 copies/mL). Both discordance and escape may be associated with neurological symptoms. We explored risk factors for CSF discordance and escape in patients presenting with diverse neurological problems.Human immunodeficiency virus type 1 (HIV-1) can replicate independently in extravascular compartments such as the central nervous system, resulting in either cerebrospinal fluid (CSF) discordance  at a single center between 2011 and 2015 were included in the analysis. Clinical and neuroimaging variables associated with CSF discordance/escape were identified using multivariate logistic regression.P = .007 and 56.9 , P = .01, respectively). All 7 patients with CSF escape (10 LPs) had been diagnosed with HIV >7 years prior to LP, and 6 of 6 patients with resistance data had documented evidence of drug-resistant virus in plasma.One hundred forty-six patients with a median age of 45.3  years underwent 163 LPs. Median CD4 count was 430  cells/µL. Twenty-four (14.7%) LPs in 22 patients showed CSF discordance, of which 10 (6.1%) LPs in 9 patients represented CSF escape. In multivariate analysis, both CSF discordance and escape were associated with diffuse white matter signal abnormalities (DWMSAs) on cranial magnetic resonance imaging (adjusted odds ratio, 10.3 , Among patients presenting with diverse neurological problems, CSF discordance or escape was observed in 15%, with treatment-experienced patients dominating the escape group. DWMSAs in HIV-infected individuals presenting with neurological problems should raise suspicion of possible CSF discordance/escape. There is a growing body of evidence that human immunodeficiency virus (HIV) can continue to replicate in disparate compartments during suppressive antiretroviral therapy (ART). Up to 10% of individuals on ART who are peripherally suppressed have detectable HIV RNA in cerebrospinal fluid (CSF) . ReasonsA clinical penetration effectiveness (CPE) score attempted to classify ART penetration into the CNS based on pharmacological properties, measurable CSF drug concentrations, and evidence of reduction in CSF viral load (VL) or improvement in cognition in response to treatment . Rawson Detectable HIV in the CSF is hypothesized to increase the risk of neurological syndromes and neurocognitive impairment (NCI) , 12, 13.Despite an increasing understanding of CSF discordance, there are gaps in knowledge, for example, identification of risk factors associated with development of CSF discordance and an understanding of which patients would benefit from further investigation in a clinical setting. In particular, data that assess the association between a patient’s neurological presenting complaint and magnetic resonance imaging (MRI) results with CSF discordance are lacking, and it is these 2 factors that most strongly lead the clinician to perform a lumbar puncture (LP). We retrospectively studied a population of HIV-infected individuals who underwent an LP for a clinical indication, to identify risk factors associated with HIV CSF/plasma discordance and CSF escape.Participants were selected using records from a single laboratory serving a population of 3800 HIV-infected individuals in 2013, the median year of study. By 2015, the number had risen to 4500, and 95% of these patients were on ART, with 90% having plasma VL <50 copies/mL. The inclusion criteria for the study were as follows: (1) adults living with HIV over the age of 18 having a clinically indicated LP at Mortimer Marker and University College London Hospital; and (2) LP performed during the period 27 October 2011 to 9 April 2015. The following LP events were excluded: (1) a repeat LP within 90 days of the first; (2) LPs where the plasma HIV VL was measured >30 days from the time of the LP, or no plasma VL was measured; (3) LPs where the MRI was done >90 days from the date of the LP.Using hospital and clinic electronic databases, data were collected on the following characteristics: age, sex, HIV VL in plasma and CSF at the time of LP (within 30 days), CD4 count within 30 days of LP, MRI brain findings within 180 days of LP, ART regimen at the time of the LP, CPE scores for the ART regimen, and clinical indication for the LP  (Supplementary Tables 1 and 2).Indications for LP were grouped into acute neurology, chronic symptoms of NCI, and subacute neurological complaint. Acute neurology was defined as new neurological presentation including headaches, confusion, sensory and motor signs, and symptoms within 1 month of onset. Chronic symptoms of NCI included global cognitive impairment, memory impairment, attention difficulties, and cognitive impairment with neuropathy, and also included follow-up consultations for a previous neurocognitive problem. The subacute neurological complaint group included any other neurological presentation (Supplementary Table 2).10 copies HIV-1 RNA greater than the VL in peripheral blood, based on criteria used in similar studies [ART regimens were divided into those containing a nucleoside reverse transcriptase inhibitor (NRTI), a nonnucleoside reverse transcriptase inhibitor (NNRTI), a ritonavir-boosted PI, an integrase inhibitor, and/or a CCR5 inhibitor . CPE scores were calculated using the scoring system devised by Letendre and colleagues . Discord studies . CSF escMRI findings were derived from clinical reports created by a specialist neuroradiologist, at the time of the patient’s presentation. The neuroradiologist was blinded to whether patients met criteria for discordance or escape. MRIs were categorized as showing volume loss, diffuse white matter signal abnormality (DWMSA), focal white matter lesion (FWML), or other abnormalities. DWMSA was further graded as subtle or definite on the basis of qualitative information provided in the report. Definite DWMSA was defined as the presence of white matter changes involving multiple regions of both supra- and infratentorial white matter . The preP value of <.1 were included in a multivariable logistic regression. A random- effects model was used to control for the effect of clustering as some participants underwent multiple LPs during the observation period. We did not perform statistical analysis for an association between ART regimen and CNS discordance/escape due to concerns regarding confounding.Statistical analyses were done using Stata software version 13 . Univariate analyses were performed to identify factors associated with discordance and CNS escape independently. Factors with a Of 194 LPs performed, 163 LPs from 146 individuals were included in the present analysis . Baseline characteristics of the study population are shown in More than one-third of patients presented with chronic symptoms indicative of NCI. The remaining patients were split fairly evenly between acute neurological presentations and subacute presentations (Supplementary Table 1). MRI reports from 141 records were available and 5 were excluded as they were performed >6 months from the date of the LP. The median time between MRI and LP was 7  days. Among LPs, 11.9% (18/151) were done in the context of focal neurological signs.P = .007)  . There wP = .003) was also associated with CSF escape in multivariate analyses  CD4 counts at the time of the LP. This observation is consistent with findings from the case series by Canestri et al . This hiWe found no association with CPE scores agreeing with the recent study by Nightingale et al , but in The limitations associated with this study mostly relate to its cross-sectional nature. The lack of association with CPE score could be confounded by potential prescriber bias. In addition, we did not measure patient compliance with ART or record the start dates of ART therapy. There is a very short decay period between when ART is initiated or changed and the CSF VL starts to fall . The indOf note, our study was not able to explore the effect of PI monotherapy on CNS discordance/escape. In the context of adequate viral load monitoring, PI monotherapy can be used in some individuals with reduced toxicity and no increase in cross-resistance , despiteIn summary, this study adds to the body of knowledge by demonstrating an association between MRI diffuse white matter signal abnormalities and both HIV discordance and escape. Previous studies of discordance and MRI findings have involved smaller numbers and have not directly measured the strength of the association. Physicians should be aware that the presence of DWMSAs on MRI in a peripherally suppressed individual with neurological symptoms may suggest HIV replication in CSF and that a normal CD4 count does not exclude this phenomenon. Longitudinal studies with serial MRI findings correlated with peripheral and CSF VL will be needed to further evaluate this association. Finally, we advocate the formation of collaborative cohorts in order to increase power to detect risk factors for virus escape in CSF under specific clinical presentations, as has been recently proposed .Supplementary DataClick here for additional data file.


Estimating muscle force by surface electromyography (sEMG) is a non-invasive and flexible way to diagnose biomechanical diseases and control assistive devices such as prosthetic hands. To estimate muscle force using sEMG, a supervised method is commonly adopted. This requires simultaneous recording of sEMG signals and muscle force measured by additional devices to tune the variables involved. However, recording the muscle force of the lost limb of an amputee is challenging, and the supervised method has limitations in this regard. Although the unsupervised method does not require muscle force recording, it suffers from low accuracy due to a lack of reference data. To achieve accurate and easy estimation of muscle force by the unsupervised method, we propose a decomposition of one-channel sEMG signals into constituent motor unit action potentials (MUAPs) in two steps: (1) learning an orthogonal basis of sEMG signals through reconstruction independent component analysis; (2) extracting spike-like MUAPs from the basis vectors. Nine healthy subjects were recruited to evaluate the accuracy of the proposed approach in estimating muscle force of the biceps brachii. The results demonstrated that the proposed approach based on decomposed MUAPs explains more than 80% of the muscle force variability recorded at an arbitrary force level, while the conventional amplitude-based approach explains only 62.3% of this variability. With the proposed approach, we were also able to achieve grip force control of a prosthetic hand, which is one of the most important clinical applications of the unsupervised method. Experiments on two trans-radial amputees indicated that the proposed approach improves the performance of the prosthetic hand in grasping everyday objects. As the basic driver of human locomotion, muscle force is an important evaluation index in research on and treatment of biomechanical diseases, disabilities, disorders, and injuries. Invasive measurement of muscle force can cause pain or injury to the subject  is commonly adopted. This approach, which assumes linear independence of the firing pattern of MUAPs , a transformed ICA with a soft form of constraint  is the firing pattern of MU j. Equation (2) indicates that the sEMG signal can be treated as a linear combination of time-shifted MUAPs. RICA and K-means were used to separate MUAPs from the sEMG signals. The details of the decomposition will be presented in the next section.where In this section, an approach to the decomposition of the sEMG signal into its constituent MUAPs and an estimation of the muscle force based on MUAPs is proposed. Figure m×n matrix to avoid singularity in decomposing the sEMG signal. In the remainder of this paper, the sEMG signal refers to data passed through this two-step preprocessing, and is denoted by x.The raw sEMG signal is sampled at 16 kHz and preprocessed in two steps. First, it is filtered by a band-pass filter  to remove noise. Then the filtered signal is standardized with zero mean and unit variance. The standardized sEMG is reshaped to a ICA is an algorithm for learning an orthogonal basis from data. However, ICA is time-consuming, because the orthogonal constraint is non-differentiable. To slightly loosen the constraint and speed up learning, the orthogonal constraint of ICA is replaced with a soft reconstruction loss and a sparsity penalty. This soft form of ICA is referred to as RICA, which reduces calculation time and is thus a practical tool for real-time data decomposition.W that minimizes the cost function J(W). The cost function of ICA constrains basis vectors to be mutually orthogonal. RICA penalizes the sparsity and minimizes the reconstruction loss, and thus guarantees that the basis vector is as orthogonal as possible  used by RICA to decompose the sEMG signal isThe model introduced in Section sEMG Model assumes that sEMG is composed of sparsely occurring and linearly independent MUAPs that are spikes in the orthogonal basis vectors of the sEMG signal. The key issue in learning the basis is to find a transform matrix 1is the absolute difference, which constrains the sparsity of the basis; and || ||2 is the square difference, which measures the differences between the original and the reconstructed sEMG signal. The optimum W can be quickly calculated with a gradient descent optimizer. x is decomposed into k basis vectors bi  by the transform matrix W:Here λ is the penalty factor (λ = 0.01); || ||bi is composed of the spike-like MUAPs and noise. The approach to detecting MUAPs from the basis vectors and clustering the MUAPs will be introduced in the next subsection.Note that An amplitude threshold is set to detect MUAPs from the basis learned with RICA. The amplitude threshold is set at 4σ to split signal from background noise:x| returns the absolute value of all elements of x. σ gives a fast and accurate estimation of background noise  are extracted from the basis vector with the peak value at sample 50. Furthermore, MUAPs with peak value less than zero are flipped to avoid misalignment of the MUAP. If the MUAP is flipped, the corresponding column in W will be multiplied by −1 to avoid changing the sign of x. MUAPs yi are extracted from the basis vector bi according to the ruleSpikes in the basis vectors sgn is the sign function, peak(bi) returns the peak of bi, and spike(peak(bi), 100) returns the 100 datapoints from bi aligned to the peak. MUAPs are clustered by the K-means algorithm in terms of their shape. The shape of an MUAP is measured according to its Fourier coefficients.The K-means algorithm minimizes the within-cluster sum of squares ] replaces 100 data points in bi from which yi has been extracted with rl, and kmeans(yi) returns the cluster label of yi. The inverse of s can be approximated by LSQR , then we assume that the MU of cluster l corresponding to the ith row of s is activated. In fact, si in c, then the MU corresponding to si is assumed to be activated. The number of activated MUs is calculated by counting the number of activated clusters. The muscle force where unique returns the number of unique elements; for example, unique = 3 owing to the duplicated 2 in the vector. For an sEMG signal in a period of 0.5 s, the muscle force is calculated by the above equation.where ϕ is a scaling coefficient and R-squared analysis:The estimated force F is the measured force, and the function var(z) returns the variance of the variable z. When R2 = 1, the estimated force explains all the variability of the measured force around its mean, and when R2 = 0, the estimated force explains none of the variability of the measured force around its mean.where To evaluate the accuracy of our force estimation approach, we carried out experiments on nine healthy subjects—seven males  and two females —after they had signed informed consent documents. The subjects reported no history of upper-extremity or other musculoskeletal disorders.The experiment setup is shown in Figure Sections Extracting MUAPs from the Learned Basis to Reconstructing sEMG Signals from MUAPs present the experimental results of the training process, and Section Accuracy of Force Estimation discusses the accuracy of the approach to force estimation during the testing process.xm×n , then bk (k = 100) basis vectors were learned from xm×n based on Equation (3). In the figure, basis vectors are colored blue and the spike-like MUAPs are colored red. One can observe that 93 MUAPs were extracted from basis vectors based on the amplitude threshold 4σ. Spikes in basis vectors of width <100 samples, marked with black ovals in the figure, were discarded owing to incompleteness. Spikes with negative peak values, which are marked with black squares, were flipped. The 93 MUAPs were clustered by the K-means algorithm into seven groups  based on their shape. MUAPs in the same cluster were highlighted in the same color, as shown on the right side of Figure The learned basis is shown in Figure Equation . In the In order to learn the basis reliably and quickly, several assumptions on the basis were made, including the sparsity of MUAPs and the linear independence of the firing patterns of the MUs. Although these assumptions have not been rigorously proved, acceptable mathematical results were obtained. Any sEMG signal in 0.05 s is expressed as a linear combination of orthogonal basis vectors that contain firing patterns of MUs in 0.05 s. In theory, for muscle force estimation, the use of large basis vectors should improve the accuracy of force estimation. On the other hand, however, large basis vectors undermine the real-time performance of the proposed approach.MUAPs of the same cluster have similar shapes, and they are assumed to be generated by the same MU. Figure Clustering of MUAPs determines the number of MUs detected in muscle. In Equation (10), the number of MUs provides the non-linearity in force estimation. Removing this non-linearity will reduce the accuracy of the proposed approach.The upper part of Figure By decomposing an sEMG signal into MUAPs, the part of the signal irrelevant to force production is removed. Reconstructing sEMG signals from MUAPs is a direct way of observing the signals after decomposition. The reconstructed signal Power spectra of the decomposed MUAPs are shown on the left of Figure R2 values of the force estimated by the proposed approach with respect to the recorded force  are 72.6, 87.3, 88.1, 72.2, 76.3, 80.2, 81.3, 84.6, and 88.9%. The R2 values of the force estimated from the amplitude with respect to the recorded force  are 67.3, 43.1, 61.3, 63.5, 57.5, 68.2, 68.9, 62.7, and 68.3%. The average R2 value of the proposed approach is 81.3% ± 6.1% and is superior to that of the approach based on the amplitude (62.3% ± 7.7%). The improvement in performance achieved with the proposed approach can probably be attributed to its robustness to superimposition of sEMG signals and to noise. From the figure, one can observe that at low force levels, where noise surpasses the sEMG signal, the approach based on the amplitude wrongly predicts a force even when none is produced. At high force levels, superimpositions of sEMG lead to severe fluctuations in the force estimated by the approach based on the amplitude, the reason for this being that the energy of the sEMG signal ceases to increase with rising muscle force.The accuracy of force estimation for the nine healthy subjects is shown in Figure The accuracy of muscle force estimation confirms the validity of the decomposition. The force can be estimated according to the biological rule stating that muscle force is proportional to the number of activated MUs and their firing rates. If the decomposed MUAPs had been inconsistent with the biological findings on MUAPs, then the muscle force estimated according to the biological rule would have diverged from the force recorded by the tensiometer. Moreover, we believe that the accuracy of the proposed approach can be improved if the parameters involved are fine-tuned for each subject.The experimental results on healthy subjects show that the proposed approach performs well in estimating muscle force. To further evaluate the performance of this approach in a clinical application, we applied it to the grip force control of our lab-made prosthetic hand.s−1 to a microcontroller that runs only Equations (9) and (10). The microcontroller thereby achieves real-time muscle force estimation.A diagram of the control strategy is shown in Figure s−1 is calculated from the sEMG signal of the MVC.An unsupervised training process is required before the amputee can use the prosthetic hand. In this training process, the amputee is instructed to perform the MVC. There are two main reasons for this: first, the MVC activates most of the MUs in a muscle and hence allows detection of a maximal number of MUAPs; second, the MVC can be used to normalize the muscle force. Grasping experiments were conducted to compare our lab-made prosthetic hand Figure  controllTwo trans-radial amputees (subject A aged 34 and subject B aged 45), after signing informed consent documents, participated in the grasping experiments. Subject A has 3 years' experience and Subject B 6 months' experience using the Kesheng hand. sEMG sensors were integrated in the socket and placed on different residual muscles of the subject. The amputees were asked to grasp everyday objects both with our lab-made prosthetic hand and with the Kesheng hand. As shown in Figure Figure We have proposed an accurate force estimation approach using one-channel sEMG and have applied it to force control of a lab-made prosthetic hand. Experimental results on nine healthy subjects indicate that this approach is more accurate than the conventional amplitude-based approach. In addition, the results of grasping experiments on two trans-radial amputees demonstrate that with the proposed approach our lab-made hand achieves more stable and faster grasping than a commercial hand. In the future, we aim to investigate the effects of segmentation interval widths of sEMG signals and of RICA redundancies on the accuracy of force estimation, as well as applying this approach to different prostheses.This study was carried out in accordance with the recommendations of the Local Ethics Committee of Peking University  with written informed consent from all subjects. All subjects gave written informed consent in accordance with the Declaration of Helsinki. The protocol was approved by the Local Ethics Committee of Peking University .WS and JZ: proposed the decomposition approach and drafted the manuscript; YJ, HY, and QH: conducted the experiments and analyzed the data.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewers YL and HW declared a shared affiliation, with no collaboration, with the authors to the handling Editor.


VMAT) planning might be jointly contributed by the patient‐specific optimization objectives, as estimated by the RapidPlan model, and by the potentially improved Photon Optimizer (PO) algorithm than the previous Progressive Resolution Optimizer (PRO) engine. As PO is mandatory for RapidPlan estimation but optional for conventional manual planning, appreciating the two optimizers may provide practical guidelines for the algorithm selection because knowledge‐based planning may not replace the current method completely in a short run. Using a previously validated dose–volume histogram (DVH) estimation model which can produce clinically acceptable plans automatically for rectal cancer patients without interactive manual adjustment, this study reoptimized 30 historically approved plans  with RapidPlan solution (PO plans). Then the PRO algorithm was utilized to optimize the plans again using the same dose–volume constraints as PO plans, where the line objectives were converted as a series of point objectives automatically (PRO plans). On the basis of comparable target dose coverage, the combined applications of new objectives and PO algorithm have significantly reduced the organs‐at‐risk (OAR) exposure by 23.49–32.72% than the clinical plans. These discrepancies have been largely preserved after substituting PRO for PO, indicating the dosimetric improvements were mostly attributable to the refined objectives. Therefore, Eclipse users of earlier versions may instantly benefit from adopting the model‐generated objectives from other RapidPlan‐equipped centers, even with PRO algorithm. However, the additional contribution made by the PO relative to PRO accounted for 1.54–3.74%, suggesting PO should be selected with priority whenever available, with or without RapidPlan solution as a purchasable package. Significantly increased monitor units were associated with the model‐generated objectives but independent from the optimizers, indicating higher modulation in these plans. As a summary, PO prevails over PRO algorithm for VMAT planning with or without knowledge‐based technique.The enhanced dosimetric performance of knowledge‐based volumetric modulated arc therapy ( The fluences are back‐projected from the derivates of the costs at each cloud point representing the patient volume. PRO optimizes MLC leaf positions and MU/deg based on control points segmentation of the gantry angle. As the optimization progresses, the accuracy of the angle resolution and dose calculation segments increase. Relative to prior optimizers, PO has involved critical changes: the old point cloud model for PRO and DVO has been replaced by a new volume representation, where “structures, DVH calculation and dose sampling are defined spatially by using one single matrix over the image”. However, DVO and PRO have a more powerful dose–volume objective form than the PO in terms of limiting the local minima in the optimization space. More technical details were described by Vanetti et al.Providing knowledge‐based planning is not likely to replace the conventional methods completely in a short run, appreciating the behavior of the new PO against the old optimizers may provide useful guidelines for algorithm selection when RapidPlan is not invoked as a separate executable option. After all, as a purchasable engine, RapidPlan is not available to every Eclipse user, and the model configuration can be a sorely time‐consuming process. In order to assess the different algorithms based on the same (but patient specific) optimization objectives that can produce clinically acceptable plans without interactive manual adjustment, all DVH constraints were derived from our RapidPlan model estimation that has been validated for rectal cancer patients.2This study was conducted based on Varian Eclipse Treatment Planning System V. 13.5.2.Aboost from GTV, and PTV from CTV, respectively). Target dose of 50.6 Gy and 41.8 Gy were prescribed to 95% of PTVboost and PTV in 22 fractions, respectively. All plans were created with 1–2 full arc, 5° collimator rotation, and 10 MV photon beams modulated by Varian Millennium 120 multi‐leaf collimator mounted on a Varian Trilogy accelerator. As reported before, the model validation on over 100 historical plans displayed significantly improved dosimetric results than the average clinical level, after applying the RapidPlan‐generated objectives and the PO algorithm.As a brief summary of the previous work,2.Bboost and PTV were covered by corresponding prescribed dose, respectively).Thirty clinical VMAT rectal plans that have been manually optimized with PRO were retrospectively selected and reoptimized using the RapidPlan engine (referred as PO plans). Representing the average planning quality, these 30 consecutively treated plans were not used for model development but followed consistent contouring2.CPTVboost and HIPTV); the target conformity index (CIPTVboost and CIPTV); dose to 50% of the volume for the femoral head and urinary bladder (D50%_FH and D50_UB), the mean dose (Dmean_FH and Dmean_UB); the hot spot volume receiving over 107% of the prescribed dose to PTVboost , and the total monitor units (MU). Based on SPSS 21 software , paired samples t‐test was used to analyze the normally distributed data (Shapiro–Wilk method); otherwise Wilcoxon signed‐rank test was conducted. Significant level was put at P < 0.05 (2‐tailed).The clinical, PO and PRO plans were evaluated mutually by means of the target homogeneity index , yet the values for PO and PRO groups were largely comparable . Relative to the clinical plans, the usage of RapidPlan‐generated optimization objectives (PO and PRO cases) has also induced: (a) Higher MUs (by 7.92% and 8.17% for PO and PRO); (b) Significant reduction of D50%_FH, Dmean_FH, D50%_UB, and Dmean_UB; (c) Significant reduction or elimination of V107%.Numerical dosimetric comparison of plans using various techniques are listed in Tables boost and PTV, respectively) was achieved by all three techniques, yet the dose to the OARs was significantly reduced using RapidPlan‐generated objectives (PO and PRO plans) than the manual settings .Figure Taking the clinical plans as baseline, Table 4boost and PTV by renormalization, PO and PRO methods have significantly lowered the target mean dose than clinical plans, indicating reduced hot spots in the targets and improved dose homogeneity  improvements of OAR sparing and hot spot control were observed after substituting the objectives from patient‐specific estimations for the trial‐and‐error manual adjustment. The objective‐induced advantages were largely transferable from PO engine to the conventional PRO algorithm, yet the performance of PRO is slightly inferior than the PO. The more adjacent DVH lines between the PO (dotted) and PRO (dashed) than the clinical (solid) plans as shown in Fig. Consistent with earlier publications,Although RapidPlan is a purchasable package, PO algorithm should be available for all Eclipse users of version 13.5 or later. Therefore, it is of patients’ maximal benefit to select PO as the preferential optimizer for VMAT planning, even if knowledge‐based solution is not purchased or the models are not yet ready (custom model configuration is treatment site‐specific and sorely time‐consuming). The slight improvement of OAR sparing by a few percent may not be clinically significant, but it complies with “as low as reasonably achievable” (ALARA) principle at little or no extra cost of effort.The lesser impact of the optimizer suggested that for the Eclipse users of older versions (before 13.5), transplanting the model‐estimated and patient‐specific objectives from other RapidPlan‐equipped centers can be a transitional option to improve the VMAT plan quality immediately even with PRO algorithm. Surely this should be performed with caution on the basis of consistent contouring/planning protocols, and thorough clinical validations.PRO−PO = 1 MU, P = 0.81). However, MUs increased considerably and significantly  after the model‐estimated objectives were introduced, indicating higher modulation in PO and PRO plans than the clinical plans. More MUs might be associated with smaller MLC apertures and lower agreement between the calculation and delivery of the intended dose distribution.The optimizers had little if any impact on the consumed MUs (ΔThis study is potentially limited by the nondeterministic nature of the optimization process: slightly different results may be obtained even if the same algorithm and constraints were utilized. However, this random and minor uncertainty should be largely canceled out by the averaging during the statistical analysis, and the systematic discrepancies should be mostly attributable to the different optimizers.5By differentiating the dosimetric improvement contributed by the knowledge‐based objectives and the new optimizer, this work suggests that PO prevails over PRO algorithm for VMAT planning, with or without RapidPlan solution.


Maxillofacial surgeries can present unique anesthetic challenges due to potentially complex anatomy and the close proximity of the patient's airway to the surgical field. Damage to the tracheal tube (TT) during maxillofacial surgery may lead to significant airway compromise. We report the management of a patient with a partially severed TT during Le Fort surgery for midfacial hypoplasia and management strategies based on peer-reviewed literature. This case illustrates the clinical clues associated with a damaged TT and explores the challenges of managing this potentially catastrophic issue.  Maxillofacial surgeries can present numerous challenges to anesthesiologists due to the potential for complex facial anatomy and the close proximity of the tracheal tube (TT) to the surgical field. Damage to the TT during maxillofacial surgery can lead to airway compromise; thus, anesthesia providers should have a strategy in place to prevent or mitigate such events. In this case, we report the intraoperative management of a patient with a partially severed nasal TT during a Le Fort surgery.A 17-year-old, 56 kg male with midface hypoplasia presented for an elective Le Fort-1 advancement surgery with bilateral malar osteotomies. His prior medical history was unremarkable. On physical examination, the patient had a Mallampati-2 airway, and his mental-hyoid distance, mouth opening, and mandibular subluxation were normal. Anesthesia was induced with sevoflurane and oxygen, obtained peripheral IV access, and applied oxymetazoline to both nares prior to smooth nasotracheal intubation with a 6.5 cuffed TT. The TT cuff was inflated with 3 mL of air; auscultation, squeezing of the pilot balloon, and palpation of the patient's neck confirmed the TT cuff's proper inflation and position.The surgeon placed a throat pack and started the procedure. While performing a left maxillotomy, the surgical team expressed concern that the TT may have been cut because of visible bubbling of gas from the nose after resection of the left lateral nasal wall. The surgeon placed the patient's head in the neutral position while the situation was assessed. The anesthesia team inspected the TT, confirmed that cephalad migration had not occurred, and discovered that the pilot balloon did not sustain inflation. The team called for help, requested additional equipment , and prepared for a possible reintubation through a bloody field. However, at this point the patient's vital signs, capnograph waveform, and the ventilator's flow-volume loop patterns had all stabilized. After a brief discussion, the intraoperative team agreed to proceed.Shortly thereafter, when the surgeons turned the patient's head away from the midline neutral position, the anesthesia machine warned of a circuit leak and air bubbles were again observed in the surgical field. The patient's head was immediately returned to the midline position and the leak again disappeared completely. The anesthesia team surmised that the tracheal tube was partially severed, resulting in an aperture that opened when the head was turned away from midline.The surgeons stated that the remainder of the case could be performed satisfactorily in the midline position. The patient's ventilation remained completely stable for several minutes, so the decision was made to proceed cautiously with the in situ TT. A tube exchange was eschewed because of the successful conservative measures and the concern of airway loss during an exchange. The remainder of the surgery was uneventful, and we extubated the patient without incident. The patient was transported safely to the postanesthesia care unit where he experienced a full recovery. Inspection of the TT revealed a cut across half of the tube's diameter; the pilot balloon tubing had been severed completely .Tracheal tube damage during maxillofacial surgery is a potentially catastrophic complication. In some cases, conservative measures such as tube stabilization and laryngeal packing provide adequate ventilatory conditions to complete a surgical case , 2. The Anesthesiologists should anticipate TT damage during maxillofacial surgery and take precautionary measures if possible. For a unilateral maxillotomy, intubation via the contralateral nares will reduce the risk of TT damage. The surgeons' use of a nasal septum osteotome with blunt horns may deflect a TT and reduce the likelihood of damage . IntraopIf damage to TT occurs during surgery, then a swift assessment of airway patency and ventilation should drive the decision to reestablish the airway. This includes examining the TT depth and auscultating the chest and direct laryngoscopy, if possible . RepositOur case illustrates the challenges of managing a damaged TT midway through a maxillotomy procedure. In our case, we chose to proceed without TT exchange due to adequate oxygenation and ventilation with the head in neutral position.


Liquorpheresis  comprises a therapeutical approach that has been proposed to treat several neurological conditions where antibodies, inflammatory mediators, or abnormal peptides are the cause or play an important role in the pathogenesis of the disease. Continuous or intermittent cerebrospinal fluid (CSF) replacement may be an alternative approach not explored thus far.Here, we review previous experiences in the use of liquorpheresis in autoimmune and degenerative neurological diseases. Then we describe previous technical reports  and provide some new innovations in order to design bidirectional CSF shunting systems that can be complemented either with a deposit of artificial CSF or with a filter of CSF, allowing CSF replacement or liquorpheresis respectively. Both options would lead to mechanical dilution of the patient’s CSF. Liquorpheresis (CSF filtration) comprises a new therapeutical approach that has been proposed to treat several neurological conditions where soluble proteins or peptides within the nervous system play an important role in the physiopathology of the disease. This is the case of several autoimmune diseases where antibodies are involved affecting the central nervous system or the nerve roots. Among them, Guillain-Barrè syndrome (GBS) is perhaps the one with more evidence supporting its use. This disorder is usually treated with infusion of intravenous immunoglobulins. Corticoids, immunosuppressants and plasma exchange are alternatives. A clinical trial tested liquorpheresis in GBS. Although the number of patients was small, it was found that liquorpheresis is at least as effective as with plasma exchange , 2. MultThe production and deposit of toxic peptides is a central fact in the physiopathology of all neurodegenerative diseases. The distinct protein aggregates that are found in neurodegenerative disorders and prion diseases seem to cause these disorders. Small intermediates (soluble oligomers) in the aggregation process can confer synaptic dysfunction, whereas large, insoluble deposits can cause cellular damage. Findings in other neurodegenerative diseases indicate that a broadly similar process of neuronal dysfunction is induced by diffusible oligomers of misfolded proteins . Then, wEven when liquorpheresis might represent a good therapeutic option in many neurological conditions, it has not been explored intensively yet, probably because it is not widely available. We devise bidirectional CSF derivation systems allowing portable liquorpheresis. Alternatively, CSF can be replaced with artificial cerebrospinal fluid (aCSF). Both options would lead to mechanical dilution of the patient’s CSF.The following paragraphs have the objective of describing previous developments and provide some new technical innovations in order to design implantable systems for continuous liquorpheresis and CSF replacement.Standard Ventriculoperitoneal DerivationVentricular-peritoneal derivation  is a method of diverting CSF from the ventricles to the abdominal cavity. It is the usual treatment of hydrocephalus, a condition produced when the natural system for draining and absorbing CSF does not work properly and the ventricles enlarge to accommodate the extra fluid. Systems for ventricular-peritoneal derivation allow draining CSF from the ventricular system  to the peritoneum. A derivation usually consists of two simple catheters (single light) and a one-way valve. The valve regulates the amount of CSF going out of the brain ventricles. As pressure increases within the brain cerebrospinal fluid, the check valve opens and excess liquid is drained to another body. There are various types of derivations. The two most common are: 1. Fixed pressure valves that regulate the amount of cerebrospinal fluid is drained based on a predetermined pressure setting. 2.  Adjustable pressure valves that regulate the amount of cerebrospinal fluid that is drained based on a pressure value which can be adjusted. Using specially designed magnetic tools, neurosurgeons may change noninvasively the pressure level of the implanted adjustable valve during a visit in consultation, without another surgical procedure. Both types of valve may include an anti-siphon device control. The purpose of an anti-siphon device control is minimize overdrainage due to the force of gravity, which can cause more cerebrospinal fluid to drain when the individual is upright. In addition, some valves include a reservoir that can be used to check the operation of the derivation and to obtain samples of cerebrospinal fluid for laboratory studies.Lumboperitoneal Derivations and Implantable Intrathecal PumpsLumboperitoneal shunting is also a method of diverting CSF to the abdominal cavity, but this one diverts CSF from the lumbar subarachnoid space instead of from the ventricles. Advantages of lumboperitoneal derivations over ventricular-peritoneal derivations include avoidance of brain penetration with the shunt catheter, access to a large CSF space in the thecal sac, and the potential of a large amount of CSF drainage. Patients who require a lumboperitoneal shunt have pathology that predisposes to obstruction of CSF absorption or elevated CSF pressure but must have communication of the spinal CSF with the cranial compartment.Implantable Intrathecal pumps are medical devices used to deliver medications directly into the lumbar subarachnoid space. They consist of a small battery-powered, programmable pump that is implanted under the subcutaneous tissue of the abdomen and connected to a small catheter tunneled to the site of spinal entry. Sophisticated drug dose regimens can be instituted. Implanted pumps need to be refilled every few months.Bidirectional Ventriculoperitoneal DerivationPatent US20090131850A1, entitled “Method and apparatus for removing harmful proteins from a mammalian's ventricular cerebrospinal fluid” devices bidirectional ventricular-peritoneal derivations complemented with a pump and a filter allowing the removal of proteins from CSF . Then, iArtificial CSF (aCSF) has been first produced in 1949 and refined since then with the purpose of using it as a vehicle solution for administration of agents to the central nervous system and neuroendoscopic surgery -9. Yang General DescriptionStandard ventricular-peritoneal   derivations does not allow diluting CSF as catheters are unidirectional. Using current technology as starting point, we envision new developments to construct implantable systems aimed at infusing either aCSF (CSF replacement) or the own patient’s CSF after filtration (liquorpheresis). This model comprises a series of interconnected elements, reservoirs, and access ports, allowing both to drain CSF to the outside  or to the peritoneum and to infuse CSF into the ventricular system  or the spinal subarachnoid space . Infusing CSF can be both aCSF (CSF replacement) and filtered CSF (liquorpheresis). Key elements in this model are the following figure :- Double lumen ventricular catheter  (one inlet and one output), which allows both draining CSF from the ventricular system and infusing fluids. As an alternative to placement in the ventricular system , the catheter can also be placed at the spinal level in the lumbar subarachnoid space .- Double valve , which allows to regulate the pressure both input and output.- Double lumen subcutaneous catheter  .- A subcutaneous injection port connected to the gateway subcutaneous catheter, which allows fluid infusion by an external pump. Additionally it can also allow access for infusing drugs with syringe if needed.  - External pump allowing continuous or intermittent infusion. It is connected to the injection port by an infusion apparatus with an L type non-coring needle.Versions and ModelsThis system may be constructed in two different versions intended for a) CSF replacement (version A)  and b) CSF filtration (version B). Thus, the pump can be complemented with a deposit of aCSF (version A) or with a filter (version B). Additionally, the system may be intended for ventricular shunting (model A) or for intrathecal shunting (model B).In version A Figure , the fluIn version B Figure  , there Basically, the difference between model A  but alternatively it can be complemented with a filter (as in patent US20090131850A1), allowing CSF replacement or liquorpheresis respectively. Both options ease the dilution of  the “dirty” CSF (with neurotoxic proteins) with “clean” CSF (without neurotoxic proteins) which can help fighting many neurological conditions.In order to maximize the dilutional effect, the system may be programed to work in an alternate mode where the drainage and infusion of clean CSF is sequentially produced to avoid the new and “clean” CSF mixing with all the “dirty” CSF in the ventricular system that would happen if the system worked in  a continuous in-out mode. Thus, some amount of “dirty” CSF should be drained before the infusion of “clean” CSF starts.The best way of programming the system has to be studied carefully to maximize the clearance effect and reduce the risk of side effects to a minimum. Side effects may appear as a consequence of producing liquoral hypotension (if too much “dirty” CSF is drained) or hypertension (if too much “clean” CSF is infused) in a given time. However, well-adjusted valves should prevent this type of malfunctioning.Other types of side effects may be related to filters and aCSF. Filters should be as specific as possible, removing only the target peptides and proteins. aCSF should have the same composition as natural CSF .  As theLiquorpheresis (CSF filtration) and replacement of CSF with aCSF comprise new therapeutical approaches that have been proposed to treat several neurological conditions. Both options have been little explored so far due to technical constraints.Developing implantable systems for liquorpheresis and CSF replacement seems suitable today from a technical point of view. We describe previous technical reports  and provide some new innovations in order to design bidirectional CSF shunting systems that can be complemented either with a deposit of artificial CSF or with a filter of CSF, allowing CSF replacement or liquorpheresis respectively. Both options would lead to mechanical dilution of the patient’s CSF.


At present, diagnosis for neurosyphilis remains a major clinical challenge. Venereal Disease Research Laboratory (VDRL) titer of the cerebrospinal fluid (CSF) is suboptimally sensitive to diagnose neurosyphilis, which can be negative in neurosyphilis patients, especially in asymptomatic neurosyphilis patients. In the search for biomarkers of neurosyphilis, we investigated the chemokine profile in CSF of neurosyphilis patients and found that the concentrations of CXCL13, CXCL10 and CXCL8 were selectively elevated in neurosyphilis patients and correlated with CSF protein concentration and CSF-VDRL titer. After antibiotic treatment, the concentration of these chemokines was dramatically reduced. The area under the ROC curve (AUC) of CSF CXCL13, CXCL8,CXCL10 and the CSF/serum ratio of CXCL13, CXCL8,CXCL10 in the diagnosis of neurosyphilis were 0.940, 0.899, 0.915, 0.963, 0.846 and 0.926, respectively. The corresponding sensitivities/specificities of CSF CXCL13, CXCL8,CXCL10 and the CSF/serum ratio of CXCL13, CXCL8,CXCL10 in diagnosis of neurosyphilis were 85.4%/89.1%, 79%/90.1% and 79.6%/91.1%, 86.6%/99%, 79%/73.3% and 86%/92.1%, respectively. Our results suggest that the elevated concentrations of CXCL13, CXCL8, and CXCL10 or their increasing CSF/serum ratios may be potential biomarkers of neurosyphilis, particularly for asymptomatic neurosyphilis. Reduced concentration of these chemokines may indicate the prognosis of antibiotic therapy. Treponema pallidum subsp. Pallidum, remains to be a global public health problem. The World Health Organization estimates that 10–12 million new infections occur each year, primarily in low and middle-income countriesT. pallidum can disseminate to the central nervous system (CNS) within days after exposure and can lead to asymptomatic meningitis that, if left untreated, can progress to severe and irreversible symptomatic neurosyphilis. Therefore, identification of sensitive biomarkers to facilitate neurosyphilis diagnosis would be valuable for preventing serious sequelae. Unfortunately, there is no “gold standard” for the diagnosis of neurosyphilis. The presence of a reactive Venereal Disease Research Laboratory (VDRL) in the cerebrospinal fluid (CSF) is specific, but the generally accepted sensitivity of CSF-VDRL is 30 to 70%33T. pallidum was isolated from cerebrospinal fluid by rabbit infection test of 12 (30%) of 40 patients with primary and secondary syphilis, but the CSF VDRL was positive in only four (33%) of these 12 patientsSyphilis, a sexually transmitted multi-stage disease caused by Chemokines and their receptors are expressed in both neuronal and glial cells of the CNS. Elevated levels of various chemokines have been found in CSF and been associated with the severity or progression of several CNS inflammatory disorders6791112In this study, we analyzed the global expression profiles of 36 chemokines in CSF of neurosyphilis patients for the purpose of screening novel biomarkers as a diagnosis of neurosyphilis in HIV-negative syphilis patients, especially in asymptomatic neurosyphilis patients.To investigate the expression profiles of chemokines in CSF of neurosyphilis patients, the levels of 36 chemokines in the CSF of 9 neurosyphilis patients  and 7 non-neurosyphilis patients  were examined using the chemokine antibody array. As shown in p = 0.000) (p = 0.008) (p = 0.000) (p = 0.013) (p = 0.000)  and non-neurosyphilis patients (n = 123). Consistent with the antibody array data, the levels of CSF CXCL13, CXCL8 and CXCL10 were 45.9 (ranged from 0.09 to 341.42), 4.3 (ranged from 0.43 to 31.96), 7.2 (ranged from 0.37 to 67.16) fold higher in neurosyphilis patients than those in non-neurosyphilis patients, respectively. The analysis demonstrated that CSF CXCL13, CXCL8 and CXCL10 concentrations did not vary significantly even after adjusting for age and sex. The CSF CXCL13 level in neurosyphilis patients  was the most significantly elevated chemokine in comparison with that in non-neurosyphilis patients  . We then= 0.008) . However= 0.000) . Of note= 0.013) . Thus, t= 0.000) .p = 0.000) (p = 0.095) between symptomatic and asymptomatic neurosyphilis patients (p = 0.006) (p = 0.004) (p = 0.000)  in comparison to that in non-neurosyphilis patients  , but thepatients . The ser= 0.006) , and was= 0.004) . The CSF= 0.000) .p = 0.000) (p = 0.008) (p = 0.003) (p = 0.000)  were significantly higher than that in non-neurosyphilis patients  . Signifi= 0.008) . On the = 0.008) , and was= 0.003) . The CSF= 0.000) .T. pallidum is capable of invading and damaging the central nervous system. CSF is abnormal in neurosyphilis patients, including a reactive CSF-VDRL, pleocytosis, and/or elevated protein concentration. Given the measurements were correlated with the disease activity, we therefore investigated the correlation between CSF CXCL13, CXCL10 and CXCL8 levels and these measurements in neurosyphilis patients. We found that CSF CXCL13 level was positively correlated with CSF protein level     (= 0.000)  and CSF = 0.000) . The CSFctively)  and CSF ctively) . There wp = 0.000). The CSF CXCL10 and CXCL8 levels were also significantly decreased. The CSF CXCL10 and CXCL8 levels before treatment were 450.3 pg/ml (ranged from 72.1 to 5603.2 pg/ml) and 100.1 pg/ml (ranged from19.5 to 442.4 pg/ml), respectively, which were reduced to 91.5 pg/ml (ranged from 31.2 to 558.7 pg/ml)) (p = 0.000) and 23.6 pg/ml (ranged from 2 to 77.6 pg/ml) (p = 0.000) after treatment, respectively. These data indicated that CSF CXCL13, CXCL10 and CXCL8 levels may serve as markers for effective antibiotic treatment for neurosyphilis patients.In 89 neurosyphilis patients who had one or more follow-up visits after treatment, CSF samples were collected after the first antibiotic treatment. As shown in p = 0.055, p = 0.050, p = 0.270, p = 0.108, respectively). The AUCs of CSF CXCL8 and the CSF/serum ratio of CXCL8 were lower than that of CSF VDRL .Given the marked elevation of CSF CXCL13, CXCL10 and CXCL8 as well as CSF/serum ratio of these chemokines in neurosyphilis patients, we further evaluated these chemokines as biomarkers in neurosyphilis diagnosis using the receiver operating characteristic (ROC) curve analysis. The optimal cut-off values were defined by the sum of maximum sensitivity and specificity, which were 256.4 pg/ml, 163.1 pg/ml, 48.1 pg/ml for CSF titer and 4.36, 1.02 and 10.3 for the CSF/serum ratio of CXCL13, CXCL10, CXCL8, respectively. As shown in p = 0.0232). The AUCs of the ratio of CSF/serum CXCL8 were lower than that of VDRL (p = 0.037). There was not statistically different between the other biomarkers and that of CSF VDRL . The specificities of the above biomarkers were close to or above 90%, except for CSF/serum ratio of CXCL8 (72.1%), as defined by these thresholds.CSF-VDRL titer is regarded as insensitive for the diagnosis of asymptomatic neurosyphilis3p = 0.0165, p < 0.0001, respectively) and for asymptomatic neurosyphilis . But it was not statistically different compared with that of CSF CXCL13 and CSF VDRL for all neurosyphilis  and for asymptomatic neurosyphilis .The combination assay of CSF CXCL13, CXCL10 and CXCL8 (Model 1) had 90.4% sensitivity, 82.9% specificity, and yielded an AUC of 0.949 (95% CI 0.926–0.972) for the diagnosis of neurosyphilis , and 77.p = 0.3968, p = 0.1386, respectively), but it was higher than that of the ratio of CSF/serum CXCL8 and CXCL10  for all neurosyphilis. For asymptomatic neurosyphilis, the AUC of Model 2 was not statistically different from that of the ratio of CSF/serum CXCL13 (p = 0.6634), but it was higher than that of CSF VDRL (p = 0.0412). Importantly, the sensitivity of Model 2 reached to 86.8%, which was higher than that of CSF VDRL (p < 0.05) for the diagnosis of asymptomatic neurosyphilis.The combination assay of the ratios of CSF/serum CXCL13, CXCL10 and CXCL8 Model 2) had 90.4% and 86.8% sensitivity, 93.7% and 94.6% specificity, and yielded the AUCs of 0.966 and 0.938 (95% CI 0.945–0.986 and 0.896–0.981) for the diagnosis of all neurosyphilis and asymptomatic neurosyphilis, respectively  and 2. T had 90.4Prompt diagnosis and treatment of neurosyphilis is important, because delayed treatment can lead to irreversible damage of CNS and inferior outcomes. Currently, the diagnosis of neurosyphilis is not difficult when patients have typical symptoms and signs. However, the diagnosis of asymptomatic neurosyphilis, especially presumptive neurosyphilis, which is mainly based on CSF abnormalities, is uncertain and difficult. Our results indicated that CXCL13 had good diagnostic accuracy for diagnosis of neurosyphilis, especially for asymptomatic neurosyphilis, which further strengthens the previous findings that CXCL13 is an excellent indicator for the diagnosis of neurosyphilis1112CXCL13 is secreted by several cell types, including B cells, monocytes, dendritic cells, nerve cells, and microglial cells14151112CXCL10 and CXCL8 are inflammatory chemokines. CXCL10 is induced by IFN-γ, which can enhance innate antimicrobial defense, chemoattracts mononuclear cells and promotes adhesion of T cells81821As the diagnostic biomarkers, the AUCs of CSF CXCL10 and CXCL8 levels and the CSF/serum CXCL10 ratio were more than or approximate to 0.9 for all untreated neurosyphilis patients, though they were lower than that of CSF VDRL. Combinatorial biomarkers can increase sensitivity or specificity7In addition, CSF VDRL may persist for years after successful treatment, whereas the levels of CSF CXCL13, CXCL10 and CXCL8 decrease rapidly after antibiotic treatment. Thus, these chemokines may be suitable for monitoring the treatment response, and differentiating an active infection from a past infection.There were some limitations in our study. First, although this was the largest and most comprehensive analysis of candidate CSF biomarkers for neurosyphilis in HIV-negative patients to date, these biomarkers are also elevated in other inflammatory diseases, such as infectious diseases and some autoimmune disorders. However, these diseases were dramatically influenced by geography, history, serological testing, and MRI findings etc., which could easily be clinically differentiated from neurosyphilis. Second, since the levels of these biomarkers could decrease dramatically after the antibiotic treatment, one must carefully analyze the results according to patients’ medication history, especially patients with probable asymptomatic neurosyphilis. Third, in patients co-infected with HIV and syphilis, the presence of CXCL13, CXCL10 and CXCL8 in CSF could be associated with syphilis, HIV or both. We were unable to determine whether the optimal cut-off values were applicable to neurosyphilis in HIV-infected patients, and the efficacy of these three biomarkers still needs to be further determined.In conclusion, our data show that CXCL13, CXCL10 and CXCL8 are potential biomarkers to be used as a complementary diagnostic tool for neurosyphilis. Similarly, they may be useful for monitoring therapeutic effect for neurosyphilis.This study was performed at the Shanghai Skin Disease Hospital between Dec. 2010 and Dec. 2014, which was approved by the Ethics Committee of the Shanghai Skin Disease Hospital. All the methods were carried out in accordance with the approved guidelines. Written Informed consent was obtained from all participants. Patients were identified and referred for enrollment by the dermatologists, neurologists, psychiatrists and ophthalmologists after careful examinations and evaluations. The diagnosis of clinical stage of syphilis was determined based on a combination of a compatible history, clinical manifestations and the results of nontreponemal and treponemal tests in the serum and CSF. The diagnosis of syphilis included a reactive serum nontreponemal and treponemal test. Exclusion criteria included age  < 18 years, traumatic CSF collection, therapeutic intervention (except serofast) and co-infected with HIV.In this study, two groups of patients were included: 1) neurosyphilis group ; 2) non-neurosyphilis group with normal CSF WBC count, CSF protein concentration and CSF-VDRL negative . In addition, for measurement of the baseline level of the chemokines in blood, 63 healthy donors, who visited Shanghai Skin Disease Hospital voluntarily for a medical check-up for the purpose of STD prevention, were recruited to the study. Fresh blood was collected and serum was isolated from these volunteers. Since it is difficult to collect CSF from healthy donors, we used a separate control group of 29 patients who underwent orthopaedic or stone surgery  but were serum RPR and TPPA negative, whose CSF samples were collected prior to spinal anaesthesia. The baseline levels of the chemokines in CSF were determined using samples from the control group. Further characteristics of the participants were presented in The diagnosis of confirmed neurosyphilis included a reactive CSF-VDRL and a reactive CSF-TPPA in the absence of substantial contamination of CSF with blood. Presumptive neurosyphilis was defined as a nonreactive CSF-VDRL but a reactive CSF-TPPA with either or both of the following: (i) CSF protein concentration > 45 mg/dl and/or CSF white blood cell (WBC) count≥8/μl in the absence of other known causes for the abnormalities; (ii) clinical neurological or psychiatric manifestations consistent with neurosyphilis without other known causes for such abnormalities24Neurosyphilis is categorized as: asymptomatic, meningovascular, paretic, ocular and tabetic neurosyphilis. Asymptomatic neurosyphilis is defined by the presence of CSF abnormalities consistent with neurosyphilis and the absence of neurological/psychiatric symptoms or signs. Meningovasculitisis defined by clinical features of meningitis and magnetic resonance image (MRI) evidence of brain lesions and/or a stroke syndrome. General paresis is characterized by personality changes, dementia and psychiatric symptoms including mania or psychosis. Patients with sensory loss, ataxia, lancinating pains, bowel and bladder dysfunction were considered as tabes dorsalis. Ocular neurosyphilis is defined by the presence of CSF abnormalities consistent with neurosyphilis and ocular signs or symptoms . Patients with all these forms of neurosyphilis have no other known causes for these clinical abnormalities.According to Chinese National STI treatment Guidelines, neurosyphilis patients were given aqueous crystalline pencillin G, 4MU intravenously every 4 h for 14 days, or ceftriaxone intravenously with 2 g daily for 10 days if allergic to penicillin. Patients returned for follow-up visits at 3, 6, 9 and 12 months after treatment. All patients underwent lumbar puncture at the 3-month visit, and lumbar punctures were repeated at 6, 9 and 12 months if the previous follow-up CSF profiles were abnormal. Blood and CSF samples were collected at each follow-up visits.CSF samples were examined using the RayBiotech Human Chemokine Antibody Array G-Series 1 , which can assess 36 chemokines in a batch including CXCL13, CCL28, CCL23, CCL27, CXCL16, CXCL5, CCL11, CCL24, CCL26, CX3CL1, CXCL6, CXCL1, CCL16, CCL1, CXCL8, CXCL10, CXCL11, XCL1, CCL2, CCL8, CCL7, CCL13, CCL22, CXCL9, CCL3, CCL4, CCL15, CCL20, CCL19, CXCL7, CCL18, CCL5, CXCL12α, CXCL12β, CCL17, CCL25. The assay was preformed according to the manufacturer’s instructions. The glass arrays were first blocked with blocking buffer and then incubated with CSF samples for 2 hours. They were then washed and incubated with biotinylated detection antibodies for another 2 hours, and subsequently with streptavidin-fluor for 2 hour. The density of image was quantified by a densitometer , and intensity was normalized with positive controls from the same glass array.CXCL13, CXCL10 and CXCL8 in CSF and serum were quantified by ELISA Kits according to manufacturer’s instructions . CXCL13, CXCL10 and CXCL8 concentration at or below the level of detection were set at the level of detection.t test.Data are presented as median and interquartile range (IQR). Differences between groups were analyzed by using Mann-Whitney U test. Spearman correlation analysis was performed between the level of CSF CXCL13, CXCL8, CXCL10 and other parameters. Differences between before and after treatment were analyzed by using paired The ability of three biomarkers to diagnosis of neurosyphilis was evaluated by receiver-operating characteristic (ROC) curves. Sensitivities, specificities, positive predictive values (PPVs), negative predictive values (NPVs) were calculated using standard formulae and expressed as percent (95% CI).We investigated the diagnostic performance of CSF CXCL13, CXCL8, CXCL10 and the ratio of CSF/serum CXCL13, CXCL10, CXCL8 for neurosyphilis diagnosis. Multivariate logistic regression analysis was applied and a model based on CSF CXCL13,CXCL10 and CXCL8 was built for neurosyphilis diagnosis, which was defined as Model1. The prediction rule was used to calculate a probability score for each patient based on their individual expression levels of the three biomarkers, where probability score =  +  + . The cut-off probability score in the combination detection assay was 1.1. According to the method as described above, we constructed a model combining the ratio of CSF/serum CXCL13, CXCL10 and CXCL8, defined as Model2, to derive a prediction rule in neurosyphilis. The probability score =  +  + . The cut-off probability score in the combination detection assay was 2.23.p < 0.05 and all reported p-values were 2-sided.Statistical analyses were performed using SPSS 17.0  and using MedCale version 8.0  for ROC curve analysis. Statistical significance in this study was set at How to cite this article: Wang, C. et al. CXCL13, CXCL10 and CXCL8 as Potential Biomarkers for the Diagnosis of Neurosyphilis Patients. Sci. Rep.6, 33569; doi: 10.1038/srep33569 (2016).


Elevated levels of donor cfDNA have been shown to correlate with solid organ rejection, but complex methodology limits implementation of this promising biomarker. We describe a cost-effective method to quantify donor cfDNA in recipient plasma using a panel of high-frequency single nucleotide polymorphisms, next-generation (semiconductor) sequencing, and a novel mixture model algorithm. In vitro, our method accurately and rapidly determined donor:recipient DNA admixture. For in vivo testing, donor cfDNA was serially quantified in an infant with a urea cycle disorder after receiving six daily infusions of donor liver cells. Donor cfDNA isolated from 1 to 2 ml of recipient plasma was detected as late as 24 weeks after infusion suggesting engraftment. The percentage of circulating donor cfDNA was also assessed in pediatric and adult heart transplant recipients undergoing routine endomyocardial biopsy with levels observed to be stable over time and generally measuring <1% in cases without moderate or severe cellular rejection. Unlike existing non-invasive methods used to define the proportion of donor cfDNA in solid organ transplant patients, our assay does not require sex mismatch, donor genotyping, or whole-genome sequencing and potentially has broad application to detect cellular engraftment or allograft injury after transplantation.Cell-free DNA (cfDNA) has significant potential in the diagnosis and monitoring of clinical conditions. However, accurately and easily distinguishing the relative proportion of DNA molecules in a mixture derived from two different sources  is challenging. In human cellular transplantation, there is currently no useable method to detect  The diagnosis of allograft rejection prompts an immediate change in management and is strongly associated with adverse outcomes. The gold standard for the detection of solid organ transplant (SOT) rejection requires tissue biopsy and histologic evaluation. However, this approach has significant limitations including the logistics of organizing the biopsy at short notice and risks associated with an invasive procedure. Non-invasive tests are safer and allow for more frequent monitoring, but current tests are generally too non-specific or insensitive for reliable rejection monitoring .13C-acetate to 13C-urea , or the targeted amplification or sequencing of proprietary polymorphic markers or genomic regions. These issues represent significant barriers to the widespread use of cfDNA as a biomarker in transplantation.Cell-free DNA (cfDNA) is released primarily during cellular apoptosis and is found in the blood of all individuals –6. In thin vitro and its utility in the first application of cfDNA to measure the presence of donor cells in a human liver cell transplant recipient and for the quantification of donor-derived cfDNA in pediatric and adult heart transplant (HT) recipients.We have developed an approach that does not require separate donor and recipient genotyping or WGS and can be completed rapidly using small volumes of plasma collected from the cellular or SOT recipient. We demonstrate our validation of this assay 9 viable human liver cells per kilogram) from a single source via a portal venous catheter for six consecutive days using a protocol identical to the CCD05 trial . Approximately 20 days after the completion of the liver cell infusions, his ammonia had normalized to less than 50 μmol/l. Peripheral blood was drawn prior to the infusion of the donor liver cells and at 1 week (1 day after the 6th infusion), 1, 3, and 6 months post-infusion. The patient was ultimately bridged to a successful liver transplant at 16 months of age.This study was approved by the Conjoint Health Research Ethics Board at The University of Calgary (study ID REB14-1244). There were two distinct clinical patient scenarios. For the first application, the patient was a male infant with X-linked ornithine transcarbamylase deficiency (OTC) due to a deletion of exons 1–10 of the OTC gene. His peak ammonia in the first week of life was 1,368 μmol/l . At 6 months of age, he received donor hepatocytes . In children, EMB was performed at 1, 6, and 12 months post-HT and then annually up to 5 years. In adults, EMB was performed weekly for 1 month starting at 2 weeks post-HT then every 2–4 weeks until 6 months and then at 9, 12, 18, and 24 months. Five tissue samples were obtained from the right ventricular surface of the septum, stained with hematoxylin and eosin, and evaluated for acute cellular rejection (ACR) using light microscopy. Tissue samples were also evaluated for the presence of antibody-mediated rejection (AMR) by C4d-positive immunohistochemical staining. Biopsy findings of ACR or AMR were reported by the responsible pathologist (not involved in the study) using standard criteria , 16. NonAfter obtaining informed consent, whole blood (2–10 ml) was collected from patients in cfDNA blood collection tubes (Streck). Blood was centrifuged at either 4,000 rpm (larger volumes) or 13,000 rpm  at 4°C for 15 min, the plasma removed, and centrifuged again at 13,000 rpm at 4°C for 15 min and the supernatant frozen at −80°C until used. Cell-free DNA was isolated from 1 to 4 ml of thawed plasma using the QIAamp Circulating Nucleic Acid kit (Qiagen). Isolated cfDNA was quantified using a Qubit fluorometer (Life Technologies), and purity (absence of nuclear DNA) was confirmed using a TapeStation 2200 D1000 tape (Agilent). In the case of contaminating genomic DNA, a size selection step was incorporated into our protocol to isolate fragmented cfDNA of 100–300 bp using magnetic SPRIselect or AMPure XP beads (Beckman Coulter).Barcoded sequencing libraries were prepared using size-selected cfDNA (1 ng) and the HID-Ion Ampliseq Identity Panel and Ampliseq Library kits (ThermoFisher Scientific). The HID-Ion Ampliseq Identity Panel that we used contained 124 SNPs on the autosomal and Y chromosomes curated from the literature –19 and dR package “mixtools” was used to generate the models  sample contributor were selected. A locus was considered homozygous if the variant was present in >86% of the sequence reads with base quality scores ≥30. Loci with >5% ambiguous base calls were excluded. Non-consensus data  at recipient homozygous loci were then collectively used to estimate the minor (“donor”) contribution through a statistical procedure known as Finite Mixture Modeling with linear constraints. For a human sample with two contributors, biology dictates that the donor model should have three non-consensus signals: 1) both alleles same as recipient 25% of the time, (2) one allele same as recipient 50% of the time, and (3) neither allele same as recipient 25% of the time , one shared allele (OSA), and no shared alleles (NSA). An initial estimate of 0.1% for the mean of the error rate was provided, based on the base quality cutoff (BAQ score of 30 = 0.1% error rate). A 1:2 ratio constraint for the means of the OSA and NSA was enforced. A 1:2 constraint for the OSA:NSA SDs was also employed. For each simulation, the final estimate of donor cfDNA contribution consists of the NSA%, minus the final error estimate. For our data, the Gaussian  distribution yielded more accurate results than the gamma distribution or other methods available in the mixtools package.To test our algorithm, HapMap DNA samples  were used to simulate a mixture of two separate populations as previously described  with samt-test was used to determine if there was a significant difference (defined as p < 0.05) between sample groups.Data are shown as means ± SD. Calculations were performed in Excel (Microsoft Corporation) for calculation of the linear correlation between the expected and measured donor gDNA concentrations, and the Student’s R2 = 0.98) correlation between the expected and measured donor DNA concentrations (data not shown) but were dismayed by the cost, inefficiency, and complex bioinformatics required for this assay. We then developed an alternative methodology that we tested using identical mixtures of HapMap gDNA. We were also able to accurately detect increasing amounts of an individual sample in a mixture and at a fraction of the cost of the WGS method (Table R2 = 0.97) zero-intercept linear correlation between the expected and measured donor DNA concentrations over three independent replicates  were often collected  in Streck tubes designed for larger blood volumes (10 ml), we were able to successfully isolate cfDNA in all cases. Different centrifugation speeds for the separation of plasma from whole blood were tested with equivalence in cfDNA yield (data not shown). Of five kits tested, greatest and most reliable yields of cfDNA from plasma were obtained using the QIAamp Circulating Nucleic Acid kit (data not shown). In our initial experiments, contaminating gDNA complicated estimates of cfDNA yield , and we added a size-selection step Figure  before pIn our infant with OTC deficiency, donor cfDNA was quantified prior to the infusion of donor hepatocytes and at serial time points post-infusion to provide a measure of engraftment success Figure . There wp = 0.02 by Student’s t-test) than background control levels  between pooled donor cfDNA levels in patients with ACR 0R (n = 15) or 1R (n = 24) and who were free of AMR  and from pediatric and adult HT recipients undergoing a clinically indicated EMB. Relatively low levels of plasma donor cfDNA were seen for all pediatric HT patients, as expected, since all patients were asymptomatic with no evidence of dysfunction by echocardiography, and none had evidence of significant ACR or AMR by EMB with all graded as ACR 0R and pAMR 0 according to ISHLT grading criteria , 16. HowR Figure .We began by replicating previous work using gDNA and WGS to detect increasing amounts of one DNA sample in a mixture . Based oThe clinical benefit of liver cell infusion as a bridge to transplantation for metabolic liver disease has been established. However, a direct, non-invasive, and clinically useful method for the detection of donor cell engraftment in the recipient is not available. Our assay provided a direct measure of donor hepatocyte cfDNA, which correlated with improved ammonia control in this patient and, therefore, likely represented material from engrafted liver cells. In our patient, we saw a rapid increase in the percentage of donor cfDNA detected at 1-week post-infusion. This may represent the early death of donor cells that do not successfully engraft and is analogous to the elevated levels of donor cfDNA seen early post-heart or lung transplantation , 21. LevIn our cohort of pediatric HT recipients, we demonstrated that low levels of donor cfDNA  can be detected in the absence of allograft dysfunction or rejection, but that these levels are distinct from non-transplant controls where the presence of “donor” cfDNA represents the error rate for base calling. Using our assay, we found that levels of donor cfDNA from adult HT recipients with either no (ACR 0R) or mild (ACR 1R) rejection do not differ significantly. This finding is consistent with the clinical practice of treating ACR 0R and 1R as equivalent and generally not altering management or increasing immunosuppression.One concern regarding the use of donor cfDNA as a biomarker is that levels of cfDNA in an individual may fluctuate over time. Changes in total circulating cfDNA have been documented after extreme exercise, trauma, or infection –28 but, a priori genotyping and complex bioinformatics. Other methods to measure cfDNA are limited by the requirement for donor–recipient sex mismatch, independent donor and recipient genotyping or proprietary components  is still required. Similarly, information collected reflects only allograft engraftment/health, and we do not obtain additional information regarding the presence of circulating viruses, which may be diagnostically useful or helpful in estimating an individual’s degree of immunosuppression , 30. Wita priori genotyping. We have demonstrated the utility of our approach in vitro and in proof-of-principle for two in vivo clinical transplant applications; the detection of cellular engraftment and the non-invasive detection of rejection. Further validation in additional transplant recipients is required, and recruitment of pediatric and adult patients is ongoing.We have described an assay that non-invasively, rapidly, and accurately quantifies the proportion of donor cfDNA in plasma from recipients of both cellular and SOTs. This assay is scalable and uses a simple algorithm that does not require donor:recipient sex mismatch or PG and SG were responsible for study design. PG wrote the algorithm. AK and SM performed the clinical hepatocyte transplant. US, NC, and VS performed the work. LD, RL, JP, RP, JW, and SW performed and optimized the sequencing. DI, US, NC, and SG recruited the heart transplant patients. SG and PG performed the data analysis. SG wrote the manuscript. All authors reviewed the manuscript.This research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. PG and SG have applied for a patent related to the described methodology.


To the editor: We wish to offer some cautionary remarks concerning the report by de Graaf et al. [f et al.  about thEurosurveillance reader was given verifiable taxonomic data about the novel Salland TBEV and its relationship with other pathogenic TBEV and (ii) a convincing degree of homology was demonstrated between the TBEV isolated from the tick and the patient. Neither of these conditions was fulfilled in this Rapid communication. Admittedly, condition (ii), although an unquestionable paradigm for zoonotic infections, will be hard to fulfil in any forthcoming TBE case because TBEV is nearly always cleared from blood and cerebrospinal fluid (CSF) already at the start of the second TBE phase, i.e. before the patient is admitted with neurologic complications [However, this case could only be called 100% waterproof, if (i) the ications ,3. Conseications , as was Thus, physicians have to rely solely on serological techniques, which have a number of flaws: TBEV shares common antigenic sites in its E protein with several other pathogenic flaviviruses, resulting in false-positive results in IgG and even IgM ELISA ,4-7. TheThere are however still other, and more obvious, question marks concerning the evidence of true TBEV infection in the case under discussion. Firstly, two titres obtained in the NT remained unchanged (1/640). Although the crucial interval in days is not exactly specified on days 24 and 36?), serological immobility is surprising for acute TBE when neutralising antibodies can be expected to increase. It is however compatible with a status of post-vaccination cross-protection. Secondly, and most importantly, CSF was only IgG-positive, while IgM was lacking. IgM-positivity in CSF is, however, paramount for diagnosing TBE and other flaviviral infections such as West Nile fever, to the extent that CSF IgG is not even considered, nor required for diagnosis, certainly not in a patient with prior contact with flaviviruses [4 and 36?Finally, CSF findings are supportive of TBE diagnosis, when there is (i) pleocytosis with predominance of segmented granulocytes over lymphocytes, (ii) impairment of the blood–CSF barrier , and (iii) intrathecal synthesis of immunoglobulins, predominantly of IgM . None ofSince CSF, for unclear reasons, remained only IgG-positive, determining the CSF/serum IgG ratio could be helpful in proving or disproving the TBE origin of this diagnostically challenging case.


In vitro and mouse model studies suggest that propofol inhibits, while isoflurane increases, neuroinflammation. If these findings translate to humans, they could be clinically important since neuroinflammation has detrimental effects on neurocognitive function in numerous disease states.Aside from direct effects on neurotransmission, inhaled and intravenous anesthetics have immunomodulatory properties. http://www.clinicaltrials.gov, identifier NCT01640275). We measured CSF levels of EGF, eotaxin, G-CSF, GM-CSF, IFN-α2, IL-1RA, IL-6, IL-7, IL-8, IL-10, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α before and 24 h after intracranial surgery in these study patients.To examine whether propofol and isoflurane differentially modulate neuroinflammation in humans, cytokines were measured in a secondary analysis of cerebrospinal fluid (CSF) samples from patients prospectively randomized to receive anesthetic maintenance with propofol vs. isoflurane (registered with n = 19) and isoflurane-treated (n = 21) patients . Increases in CSF IL-6, IL-8, IP-10, and MCP-1 levels directly correlated with each other and with postoperative CSF elevations in tau, a neural injury biomarker. We observed CSF cytokine increases up to 10-fold higher after intracranial surgery than previously reported after other types of surgery.After Bonferroni correction for multiple comparisons, we found significant increases from before to 24 h after surgery in G-CSF, IL-10, IL-1RA, IL-6, IL-8, IP-10, MCP-1, MIP-1α, MIP-1β, and TNF-α. However, we found no difference in cytokine levels at baseline or 24 h after surgery between propofol- (These data clarify the magnitude of neuroinflammation after intracranial surgery, and raise the possibility that a coordinated neuroinflammatory response may play a role in neural injury after surgery. Patients were excluded if they were (1) unable to provide informed consent, (2) <18 years old, (3) pregnant, (4) imprisoned, or (5) had a personal or family history of malignant hyperthermia or other medical contraindication to receiving propofol or isoflurane. The primary aim of the parent study was to evaluate the effect of anesthetic type on CSF AD biomarkers, such as amyloid beta, tau, and phosphorylated tau  trial . MAD-PIATo determine the impact of surgical procedure type on neuroinflammation, surgical cases were divided into three types Table : 1, peri® catheter at the L4–L5 or L5–S1 interspace, and then connected it to an external CSF drain .Patients were randomized to receive either propofol or isoflurane for anesthetic maintenance; however, all patients received propofol for anesthesia induction. Depending on the patient’s randomization assignment, anesthesiologists (and anesthesia residents and nurse anesthetists) were instructed to titrate propofol or isoflurane dosage to maintain a bispectral index (BIS) value of 40–60. There were no protocol restrictions regarding the use of other anesthetic drugs, such as opioids, paralytics, or steroids. After anesthetic induction but before surgical incision, a member of the surgery team inserted a subdural SilasticFresh CSF samples (10 ml) were obtained from the lumbar drain at the time of drain placement (0 h) and 24 h later, and placed in a 15 ml conical tube  on ice. The CSF samples were divided into 1 ml aliquots in pre-chilled Sarstedt 1.5 ml polypropylene microcentrifuge tubes  with low-binding 1,000 µl pipette tips  and then stored at −80°C.A wide variety of molecules and cells are involved in various human inflammatory disease states –24. Sinca priori sample size calculation. To estimate the potential effect size of anesthetic type upon human postoperative CSF cytokine increases after intracranial surgery, we measured cytokine levels in CSF samples from patients in the MAD-PIA study (n = 19) vs. isoflurane (n = 21) for anesthetic maintenance.This study aimed to test the hypothesis that propofol and isoflurane have differential effects on postoperative human neuroinflammation. To the best of our knowledge, no prior randomized human study has measured CSF cytokine levels before and after surgery in patients who received propofol vs. isoflurane. Thus, we judged there to be insufficient preliminary data to perform a formal IA study , who wert-tests for continuous variables and chi-square tests for categorical variables in Table p values [Table Baseline and intraoperative differences between the propofol and isoflurane treatment groups were evaluated with Study enrollment is depicted in Figure Cytokine levels were measured in CSF samples obtained before and 24 h after surgery. Even after Bonferroni correction , G-CSF, To determine whether postoperative changes in CSF cytokines were correlated with one another, we calculated spearman correlation coefficients between each two-way cytokine combination . Roughly 40% (or 46) of these 120 possible two-way cytokine elevations were positively correlated with one another Figure . Of thesp > 0.05 for each cytokine, after Bonferroni correction  as those seen here have been reported in both AD , 29 and vice versa, prior studies have clearly shown that neuroinflammation impairs neurocognitive function at multiple levels ranging from synaptic transmission  directly correlated with one another and with CSF tau increases, consistent with a coordinated postoperative neuroinflammatory response. Future studies are needed to further characterize this postoperative neuroinflammatory response and its clinical implications.This study was carried out with approval from and in accordance with the regulations of the Duke University Medical Center Institutional Review Board. All subjects gave written informed consent in accordance with the Declaration of Helsinki, before any study activities began. The protocol was approved by the Duke University Medical Center Institutional Review Board.MB conceived of, designed, and oversaw this study, and organized the manuscript. VP and MB wrote the manuscript. JN, MB, AF, DM, DW, and MJ helped enroll patients and obtain samples. MC helped in data extraction and design of statistical analysis, NG helped in design of and performed statistical analysis. LS helped perform and oversee assay measurements. JM, MN, DM, and MJ helped design and oversee the study. All authors made substantial contributions to this work, helped revise the manuscript, approved the final manuscript, and agreed to be held accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.MB acknowledges research funding from Minnetronix, Inc., for a study unrelated to the topic of this manuscript, material support from Massimo for another study unrelated to the topic of this manuscript, and private consulting income for a legal case related to postoperative cognition in an older adult. The rest of the authors have no other conflicts to report. The reviewer RL declared a shared affiliation, with no collaboration, with one of the authors, LS, to the handling editor.