The aptasensor responded to RBP4 in the linear concentration range from 0.001 to 2 ng/mL and detection limit was slightly less than 1 pg/mL. The proposed method has successfully applied to determine the RBP4 in patient real serums. By using the intercross-linked gold nanoparticles, it is possible to provide more accessible surface for immobilizing luminol and enhance the chemiluminescence signal. Therefore, the analytical parameters such as sensitivity, specificity, detection limit and linear range were improved in compare to the biosensors reported in the literature.Prognosis of diabetes risk at early stages has become an important challenge due to the prevalence of this disease. Retinol binding protein 4 (RBP4), a recently identified adipokine, has been introduced as a predictor for the onset of diabetes type 2 in coming future. In the present report a sensitive aptasensor for detection of RBP4 is introduced. The immune sandwich was prepared by immobilizing biotinylated RBP4 aptamers on streptavidin coated polystyrene micro-wells and then incubation of RBP4 as target and finally addition of luminol-antibody bearing intercross-linked gold nanoparticles as reporter. The chemiluminescence intensity was recorded in the presence of hydrogen peroxide as oxidant agent and Au T2D can lead to many serious complications including cardiovascular, kidney and eye diseases and nerve damages as well. These diseases eventually may lead to premature death4. Moreover, treatment of T2D and its complications cost billion dollars per year for health care and also indirect costs caused by loss of productivity from disability and early mortality5. Accordingly, preventing or delaying the diabetes will improve the quality of individual life via reducing the diabetes-related complications. In recent years several various gene and protein biomarkers including retinol binding protein 4 (RBP4) have been introduced for prognosis of T2D6. RBP4 has been introduced as an independent predictive biomarker at early stages of insulin resistance7. The blood serum levels of RBP4 correlates significantly with other major factors related to insulin resistance such as triglyceride, cholesterol and blood pressure. Explaining the mechanism for the relation of elevated RBP4 with the risk of insulin resistance and T2D is complex due to the dark aspects of this process8. High fat storage in adipose tissues elevates the levels of RBP4 by which the expression of the insulin-stimulated glucose-transporter4 (GLUT4) in adipocytes is selectively reduced and consequently leads to decrease glucose uptake. In addition, increasing the level of RBP4 in blood serum might contribute to impair insulin-stimulated glucose uptake in muscle by decreasing enzyme PI-3 kinase activity. However, in liver, it can elevate hepatic glucose production by stimulating phosphoenol pyruvate carboxy kinase expression which is the characteristics of T2D9. An early study in 2014 has shown that RBP4 directly induces macrophages to secrete proinflammatory cytokines which results in greater inflammation in adipose tissue and insulin resistance10. Thus, these various effects of elevated level of RBP4 can lead to insulin resistance before any significant changes in diabetic markers in high risk population11. So, the elevation of RBP4 level can be a sign for catching the diabetes.Type 2 diabetes (T2D) has become a major challenge for public health worldwide. Increasing the prevalence of T2D could be due to the factors such as aging, urbanization, obesity, physical inactivity and modern lifestyle. Among them, obesity contributes in insulin resistance and abnormal glucose metabolism12. However, for improving specific detection and precise quantification of RBP4, aptamers were used. Aptamers are synthetic single-stranded oligonucleotides with high specificity and affinity to their targets that make them ideal candidates for molecular recognition systems13. Aptamers have been applied in biosensor design (aptasensor) due to their advantages over antibodies including: stability, availability, modifiability, flexibility and in some cases better specificity and affinity than similar antibodies14. RBP4 binding aptamer (RBA) is a 76-bp ssDNA sequence with very low Kd value (0.2 ± 0.03 µM)15. The specificity of this aptamer (RBA) has already been confirmed by using Vaspin, Nampt, adiponectin (ADPN), human serum albumin (HSA), human IgG and fibrinogen as negative controls16. RBA has also been applied in the enzyme-linked antibody-aptamer sandwich system with colorimetric reaction for more convenient and sensitive detection of RBP4 (78 ng/mL)16.So far, various methods including ELISA (enzyme-linked immunosorbent assay) and conventional antibody-based assays have been presented for the quantitative measurement of human RBP4In the present study, an ultrasensitive aptasensor has been designed for the assessment of human RBP4 level in blood serum. In this system, RBP4 has been captured by immobilized RBA on plate and then detected by polyclonal luminol-antibody bearing intercross-linked gold nanoparticles (GNPs). GNPs were intercross-linked to each other via cysteine as a simple and low cost linker. Such big clusters increased the accessible surface for conjugating polyclonal antibodies and luminol molecules. Consequently, the efficient conjugation of polyclonal antibodies increased the probability of antibody-antigen interaction and on the other hand, the proficient conjugation of luminol molecules on GNP clusters could amplify the chemiluminescence signals. To our knowledge, there is no report applying RBA in chemiluminescent aptasensor. Application of chemiluminescence as a highly sensitive physical transducer together with the luminol-antibody bearing intercross-linked GNPs, will level up the aptasensor signal to an ultrasensitive scale.For single and intercross-linked GNPs characterization, the nanoparticle suspension was diluted in deionized water. The diameter of diluted single GNPs was measured by DLS to be in the range from 18 to 28 nm, while that for intercross-linked GNPs was in the range from 28 to 40 nm. These values were also confirmed by the images obtained by transmission electron microscopy Fig. .Figure 117:570 calculated using the Eq. 17, the single and intercross-linked GNPs concentrations were estimated to be 1.56 × 10−6 and 1.7 × 10−9 M, respectively:The extinction coefficient (ε) for GNPs was calculated according to Eq. 1entclass1pt{minima18. By immobilization of antibody and luminol on GNPs, the UV-Vis absorption peak was even shifted in some extend.Intercross-linking of GNPs was also confirmed by using UV-Vis spectroscopy. In Fig. −1, one may conclude that the free -SH groups in MUA and cysteine have already been bound onto GNPs surface. The characteristic peaks of –COOH vibrations at 1725 cm−1 and the saturated alkyl stretching vibration at 2867 and 2918 cm−1 were also indicated the presence of MUA onto GNPs surface. The peak at 1641 cm−1 (C=O stretching amides), the signal at 3400 cm−1 (–NH stretching amide) and the peak at 1350 cm−1 (C–N) prove the formation of (C–NH) amide bond via reaction between the primary amine groups of cysteine and the carboxylic groups of the MUA. The peaks at 760 and 1200 cm−1 correspond to the vibration of C–S bond which confirm the cysteine conjugation. These results indicate the formation of GNPs intercross-linked.ATR-FTIR Spectroscopy was performed to characterize the intercross-linkage of GNPs. Since no characteristic absorption peak was observed for free -SH at 2559 cmThe ability of the aptasensor for RBP detection in a wide range of pH in PBS and carbonate buffers was investigated while the chemiluminescence signals were measured. The obtained data showed that the aptasensor reached to its maximum working sensitivity in 0.02 M PBS buffer at pH 8.0.To optimize ionic strength, the sensor response was recorded in PBS containing different NaCl concentrations . The maximum signal was obtained in 0.02 M PBS (pH 8) containing 100 mM NaCl.In order to assess the ability of intercross-linked GNPs in signal amplification, the chemiluminescence intensity resulted from luminol-antibody bearing GNPs was compared with that obtained from luminol-antibody bearing intercross-linked GNPs. As shown in Fig. 19. But in the present study, GNPs were intercross-linked to each other via Cys as a simple and low cost linker  solution during 21 days while the solution was kept at 4 °C in dark. Comparison of the chemiluminescence signals revealed that 95% of the initial intensity was retained after a 21-day storage period.16.Figure 23.Under the optimized conditions, the calibration curve for determination of RBP4 was plotted. As shown in Fig. The feasibility of present aptasensor for real samples was assessed by 20 serum samples from normal and diabetic individuals, obtained from Tehran University of Medical Sciences. At the same time RBP4 in real samples was determined by the aptasensor and Human ELISA Kit (ab108897) and the results were compared in Tables In addition, the experimental course for conventional ELISA takes about 3 hrs, while using the present biosensor this process would be don rapidly. Because in this approach the luminol-antibody bearing intercross-linked GNPs is prepared before starting the measurement. Moreover a simple and inexpensive laminator is used as detector of biosensor that record signals quickly. These features demonstrate the efficiency of biosensor for measuring RBP4 in real patient serums. Therefore, it seems that it has the potential to be applicable for commercial purposes.11. In the present study, an ultrasensitive aptasensor for monitoring RBP4 at the detection limit of 951 fg/mL was designed and applied for predicting diabetes at early stages. Cysteine as an available, low-cost and easy to operate linker was used for intercross-linking of GNPs. By using the intercross-linked GNPs, it was possible to provide more accessible surface for immobilizing luminol and consequently amplifying the signal. Since the luminol-antibody bearing intercross-linked GNPs was prepared before starting the measurement, the aptasensor operation became relatively fast in comparison of the most previously reported detection systems. Detection of RBP4 was carried out by formation of an immune complex via a heterogeneous interaction between aptamer-RBP4-antibody which increases the specificity of sensor25. Moreover, a simple and inexpensive laminator was used as detector of biosensor that record signals quickly. As seen in Table 26. Therefore, the features demonstrated in Table Obesity and accumulative fat storage increase secretion of RBP4 from adipose tissues and plasma concentration. Elevating the level of RBP4 could be a sign for early detection of T2D2O2 (30% solution) and three sodium citrate (Na3C6H5O7) were purchased from Merck (Germany). Gold (III) chloride hydrate (HAuCl4), bovine serum albumin (BSA), human serum albumin (HSA), Fibrinogen from human plasma, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS), 11-mercapto undecanoic acid (MUA), 2-morpholinoethansulfonic acid monohydrate (MES), nonionic surfactant polyoxyethylene-20 sorbitanmonolaurate (Tween-20) and absolute ethanol were purchased from Sigma-Aldrich. RBA  modified with biotin-T10 (10 times repeated thymidine bases) were provided by Metabion Inc., . The sequence of RBA16 was: 5′-Biotin-TTTTTTTTTTATACCAGCTTATTCAATTACAGTAGTGAGGGGTCCGTCGTGGGGTAGTTGGGTC GTGGAGATAGTAAGTGCAATCT-3′. Human RBP4 protein (ab114060), anti-RBP4 antibody (ab114062) and RBP4 Human ELISA Kit (ab108897) were purchased from Abcam  and the streptavidin coated polystyrene micro well were provided by Pishtaz Teb Zaman Co. . All chemicals used without further purification. Also in all experiments the solutions were prepared using double distilled deionized water. All the instruments were set to zero with a blank solution to eliminate the interferer substances. A solution containing 150 mM NaCl, 25 mM Tris, 0.1% BSA and 0.05% Tween-20, pH 7.2, was prepared and used as washing solution. Another solution containing 100 mM NaCl, 20 mM Tris–HCl, 2 mM MgCl2, 5 mM KCl, 1 mM CaCl2 and 0.02% Tween-20, pH 7.6, was prepared and used as binding buffer.Luminol , HDynamic light scattering  was used for size determination. The single and intercross-linked GNPs images were obtained by PHILIPS CM30 Scanning Transmission Electron Microscope . For UV-Visible spectroscopy, the Cary 100 bio spectrophotometer  was used. Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR- FTIR) was performed using Nicolet Nexus 470 FTIR  spectrometer. IrAnalyze software was also applied for IR spectrum interpretation . TMicro centrifuge  was used to separate GNPs at different conditions. The samples were centrifuged at 14000 rpm for 30 min at 4 °C. The chemiluminescence emissions were recorded at 425 nm by fluorescence micro plate reader .27. Based on the addition of different volume of 1% tri-sodium citrate to HAuCl4 solution, GNPs (Φ: 20–25 nm) were prepared. The resulting particle suspension stocked in a brown bottle at 4 °C.GNPs were synthesized according to reference No.As shown in Fig. During first process, carboxylated GNPs were prepared in such a way that 1 mL of GNPs was gently added to 2 mL of phosphate buffer solution  containing MUA (3 mM) and Tween-20 (0.2 mg/mL) Fig. . The mix28, cysteine was self-assembled on GNPs  as catalyst and H2O2  as initiator were injected to the solution and the chemiluminescence intensity was recorded. Then calibration curve was plotted based on chemiluminescence intensities against the serial dilution of RBP4 protein. Then detection limit (DL) was calculated according to Eq. 329:a is the standard deviation of the response and b is the slope of the calibration curve.The response of aptasensor in PBS containing different concentrations of NaCl were investigated. For chemiluminescence measurements, optimum PBS buffer  was added to the immune complex and then HAuCl30:The feasibility of present aptasensor for real samples was assessed by 20 serum samples from normal and diabetic individuals, obtained from Tehran University of Medical Sciences. At the same time RBP4 in real samples was determined by the aptasensor and Human ELISA Kit (ab108897) and the results were compared. To verify the reproducibility of responses, the coefficient of variation (%CV) was calculated. First, the standard deviation (σ) was calculated according to Eq. 431:To analyze intra-assay CV, three replicates of three different samples were run in one assay. To analyze inter-assay CV, three different samples were run in three independent assays. The inter or intra-assay variation (%CVs) was calculated using Eq. 5The values of %CVs less than 10 and 15, respectively for intra-assay and inter-assay, are generally acceptable.This study complied with the Declaration of Helsinki (1989 revision) and was approved by the Ethics Committee of the University of Tehran. All of the participants signed an informed consent form before enrollment in the study. All methods and experiments were performed in accordance with the standard ethical guidelines and approved by Ethics Committee of the University of Tehran.


Following this, the individual input factors of the KDPI were analyzed to assess their potential to evaluate the quality of a donor organ. We found that a donor’s age alone is significantly predictive in terms of 1-y-eGFR and death-censored allograft survival (both p < 0.001). Therefore, a donor’s age may serve as a simple reference for future graft function. Furthermore, we found that an organ with a low KDPI or from a young donor has an improved graft survival rate whereas kidneys with a high KDPI or from an older donor yield an inferior performance, but they are still acceptable. Therefore, we would not encourage defining a distinct KDPI cut-off in the decision-making process of accepting or declining a kidney graft.The Kidney Donor Profile Index (KDPI) was introduced in the United States in 2014 to guide the decision making of clinicians with respect to accepting or declining a donated kidney. To evaluate whether the KDPI can be applied to a European cohort, we retrospectively assessed 580 adult patients who underwent renal transplantation (brain-dead donors) between January 2007 and December 2014 at our center and compared their KDPIs with their short- and long-term outcomes. This led to the observation of two associations: one between the KDPI and the estimated glomerular filtration rate at one year (1-y-eGFR) and the other between the KDPI and the death-censored allograft survival rate (both  Moreover, there is no evidence that histological assessment, may serve as an independent predictor of allograft survival unless there is readily accessible donor data13.While choosing a recipient, a critical issue araises in terms of evaluating the kidney graft quality. Several methods and scores have been employed to evaluate organ’s quality which mainly depend on clinical and, in part, histological donor data. However, some of these methods assess other recipient parameters as well14.Furthermore, in the context of Eurotransplant, procurement biopsies are not routinely performed as the main focus is placed on the evidence-based objective of short ischemia timeset al. identified advanced donor age as a leading determinant of reduced allograft survival15. Further studies have confirmed the validity of this finding and have associated it with the loss of functional nephrons with age17. Even in the standard criteria donor/extended criteria donor (SCD/ECD)-dichotomy, established in the United States (US) in 2002, age plays a decisive role as well18. Unfortunately, the ECD/SCD labeling has been proven to insufficiently predict the prognosis, as kidneys are suspected to be discarded unnecessarily due to the stigmatization that arises from the ECD/SCD label21.During the mid-90’s, Chertow 22.At present, the Kidney Donor Profile Index (KDPI), based on the Kidney Donor Risk Index (KDRI), has been established as the most effective scoring system in the US, in terms of assessing the individual risk of a deceased donor’s kidney23.The KDPI is a percentile measure that is based on the United Network of Organ Sharing’s (UNOS) data from The Scientific Registry of Trasplant Recipients (SRTR). It examines the risk of kidney failure for a given donor relative to the risk for an average kidney donor from the previous year. The KDPI has been implemented as the central component of a new kidney allocation system that was adopted by the US in 2014, which is basically a longevity matching allocation . It replaced the former SCD/ECD-dichotomy to optimize the utilization of kidneys and prohibit unnecessary discards22.Calculation of the KDPI includes ten variably weighted donor parameters that significantly influence donors’ organ quality with particular focus on nephron mass: age, height, weight, cause of death, last serum creatinine, history of diabetes, hypertension, HCV-infection, ethnicity, and the discrimination between donation after brain death versus donation after cardiac deathOf all the variables listed above, age has the highest negative impact in the KDPI algorithm, especially if a donor is above 50 years or below 18 years. Furthermore, the KDPI increases, when the donor is shorter and lighter than 80 kg or shows an increased serum creatinine level.In Germany there is no valid stratification score or tool to assist physicians in their decision of accepting or declining an organ offer. However, as the data required to calculate the KDPI is available in Germany, we evaluated the KDPI using retrospective data from our transplant center. The results were interpreted by taking into account that the Eurotransplant allocation system is fundamentally different from the US one and that German conditions with respect to health care system, population, and geographics differ from those in the US.We conducted a retrospective single-center cohort study of 580 adult patients who received a deceased donor renal transplantation (RTx) at the University Hospital of Münster, Germany between January 2007 and December 2014. Recipients younger than 18 years as well as living donor recipients were excluded , and the methods adopted by this study were conducted in accordance with the current transplantation guidelines and the Declarations of Istanbul and Helsinki. Furthermore, written informed consent was obtained from all participants at the time of transplantation permitting recording and using their clinical data for scientific research. As donors were braindead at the time of donation and as this study is a retrospective, non-interventional study, written informed consent of donors was waived. Organ donors had previously expressed a wish to donate their organs in legal terms. No samples were procured from prisoners. Organs were procured on behalf of Eurotransplant and the Deutsche Stiftung Organtransplantation (DSO) in different hospitals of the Eurotransplant area. Transplantations were performed by the Department of General, Visceral and Transplantation Surgery at the University Clinics Münster, Germany.To display the unexceptional transplant population and contracted by the limitations of retrospective considerations, no further inclusion- or exclusion criteria were implemented.22.Donor data was obtained from the Eurotransplant Network Information System (ENIS) and the KDRI and KDPI were assessed using the Organ Procurement and Transplantation Network (OPTN) online calculatorIn case of missing information on the history of diabetes or hypertension data was substituted by mean values according to the given algorithm . Recipient and outcome parameters at baseline and during follow-up were retrospectively collected from the patients’ files. The recipients were regularly revisited within the scheduled out-patient aftercare and treated in-patient in case of complications.24.The set of covariables used in our study is based on theoretical considerations. We selected common parameters that have been established by numerous studies over a long time period which investigated outcome and risk factors in renal transplantationThese confounders have been chosen to cover the fundamental categories of outcome related pre- and peritransplant parameters that are not included in the KDPI: basically physical and medical recipient characteristics, procedural aspects and immunological conditions. However, the number of included variables has to be limited to a number which is appropriate for our sample size.27.Primary outcome measure was the estimated glomerular filtration rate (eGFR) at one year after transplantation (1-y-eGFR) pertaining to patients alive at this point of time, as the quantitative graft function at one year has been established as an appropriate indicator for long term transplant successThe eGFR was calculated using the CKD-EPI formula and was based on a whole-blood analysis for creatinine .Patient survival was defined as the time from RTx to death (from any cause) or last contact with alive patients. Death-censored graft survival was defined as the time from RTx to graft failure where death without prior graft failure was regarded as censored.Secondarily, we assessed the incidences of delayed graft function (DGF), defined as need of dialysis within the first week post-transplantation, frequencies of biopsy-proven acute rejections (AR) within the first year and rates of complications requiring surgical intervention, also within the first year.Following a preliminary series of univariate analyses for the chosen set of potential covariates . Continuous variables are shown as the median ± interquartile range (IQR) and the absolute and relative frequencies were provided for categorical variables.Multivariable linear regression was conducted to determine independent factors that influence the 1-y-eGFR. We included the following recipient- and procedure-specific variables which, according to the aforementioned preconsiderations and preliminary univariate analyses, can potentially impact on the graft’s function: age, sex, body mass index (BMI), dialysis vintage (DV), CIT of the donor organ, condition of previous RTx, human leucocyte antigen (HLA) -matching and current panel reactive antibodies (cPRA%).The results were presented as regression coefficients (B) with 95%-confidence intervals.To estimate the probability of DGF, acute rejections, and surgical complications multivariable binary logistic regression analyses were conducted. Therefore, a multivariable model was built using a stepwise variable selection procedure . The variables included age, sex, BMI, time on dialysis, CIT, condition of previous RTx, HLA-matching, and cPRA%. The results were presented as odds ratios (OR) with 95% confidence interval (95% CI) and p-value of a likelihood-ratio test. For the non-selected variables in the multivariable analyses, the p-value of the score test was provided.28, and KDPI- and donor’s age-groups were compared using log-rank tests. Cox proportional hazards regression models29 were built using a stepwise variable selection procedure to assess the association between the KDPI and patient and allograft survival while simultaneously adjusting for potential confounding factors . The results were presented as hazard ratios (HR) with 95% confidence interval (95% CI) and the p-value ofa likelihood-ratio test. For the non-selected variables in the multivariable analyses, p-value of the score test is given.Patient and death-censored allograft survival (based on the assumption that death is unrelated to the transplant) were estimated using the Kaplan-Meier methodAnalogous analyses were also conducted by including the aforementioned recipient- and procedure-specific variables, but instead of considering the fully computed KDPI each of its input parameter  were taken as individual variables to examine their discrete predictive value with respect to the examined outcomes (Model 2), which were again adjusted to the same selection of covariates.The correlation between the KDPI and donor age was investigated by assessing Pearson’s and Spearman’s coefficients of correlation.No adjustments were made for multiple testing and the analyses were regarded as explorative. P-values < 0.05 were considered as statistically noticeable.Tables In our cohort of 580 kidney transplant recipients the median KDPI was 71% (IQR: 47–91%) .The KDPI increased in parallel with the donors’ last creatinine level. Ten percent of donors were diabetics; approximately one third had a history of hypertension and two-thirds died due to a cerebrovascular event.2. Furthermore, the 1-y-eGFR was found to decrease as the KDPI range increased  revealed a noticeable association between KDPI and 1-y-eGFR. Further variables that were associated with 1-y-eGFR included a higher recipient BMI, a longer CIT and a higher number of HLA-mismatches. The analogous analysis was conducted after adjustments for each KDPI input parameter (Model 2). Here, we found donor age to significantly influence 1-y-eGFR. Additional donor factors that correlated with 1-y-eGFR included the last serum creatinine and a cerebrovascular cause of death – in fact, these were two of the three ECD-criteria Table .Table 3MFor the total time of follow-up 63 patients suffered from total graft failure (11%) and 47 died with functioning graft (8.1%). 13 (2.2%) patients died after graft failure due to other reasons.Kaplan-Meier curves for death-censored allograft survival by KDPI category are shown in Fig. No difference was found between the groups in terms of patient survival, neither when analyzed by KDPI nor by donor age category  and the donor age (as the leading parameter of Model 2) but no other covariates to create an unbiased comparison. Both parameters were entered into the analysis without applying any method of selection. Herein the KDPI (p < 0.001) was found to be marginally superior to donor age (p 0.004)  -analyses were conducted to evaluate the performance of the KDPI, donor age, and ECD/SCD in terms of predicting allograft failure , whereas a kidney with a KDPI > 85% does not accomplish half of this periodHowever, the question remains whether this discrepancy arises due to the fact that “inferior” organs are selected for “sicker” patients.33.Several studies have been conducted among American and European populations to evaluate the validity of the KDPI to determine a dontation’s quality34. Lee and Abramowicz from Antwerp in Belgium anticipated the KDPI in 2015 to become an advantageous tool to qualitatively evaluate kidney donations across Europe3. After completing their own investigations in 2017, they confirmed that the KDPI provides good guidance regarding whether to accept or decline an organ offer, and they encourage other European centers to validate this score35. The most recent studies in the field were conducted in Berlin, which demonstrated the utility of the KDPI in terms of donor quality assessment in a German cohort36, and from Barcelona. Both studies still demand more data before suggesting the use of the KDPI in clinics37. The present study confirms the applicability of the KDPI in a kidney transplant cohort of Münster, Germany, showing it has good predictive power in terms of 1-y-eGFR and death-censored allograft survival. Our median KDPI matches quite well with those from Berlin36 and Barcelona37 and thus appears to represent the European donor population.A Canadian paper from 2017 has highlighted the need to determine the applicability of KDPI in different regions, as its application did not seem to improve the prediction of allograft failure in a British Columbian cohort in comparison with the simple criterion of donor ageWe notably found that an improved graft survival of an organ with a low KDPI is contrasted by a longer waiting time . One explanation for this might be that older organs (with higher KDPI) are often allocated by the Eurotransplant algorithm of the European Senior Programm (ESP), which aims to shorten waiting time for patients older than 65 years. In this regard, we observed an impaired but still acceptable outcome of kidneys with a KDPI > 85%. The eGFR was approximately 10 ml/min below total average (40 vs. 50 ml/min), but total graft failure was observed in only 13.7% vs. 8.6% of cases. As the waiting time for these organs was found to be 16 months less on average, we would advise transplant physicians to not generally decline an organ donor just because of a KDPI > 85%.et al. who found significantly worse but acceptable outcomes of high and even very high KDPI kidneys as well  and also emphasized the benefits of the ESP program in this context36.This result is consistent with the results of Lehner In summary, the KDPI may be a supportive tool in objectifiying clinical evaluations and ranking the prognosis to some extent in clinican’s decision-making process.22. Therefore, according to our data the use of a distinct KDPI cut-off cannot be recommended.The general predictive power of the KDPI is moderate. In the original trial the c-statistics was 0.6, which means that outcomes for organs with strongly different KDPI values can be prognosed more precisely than for organs with similar KDPI valuesThe correlation between donor age alone and our leading outcomes was highly significant as well, which is not surprising, given that both parameters were closely correlated.The KDPI may be marginally superior, as it takes creatinine level and cerebrovascular cause of death into account, which prove to be of additional significance concerning our primary outcome measure. This assumption is supported by the results of the aforementioned bivariate linear regression analysis shown in the Supplementary Table With respect to the other KDPI input factors, we did not observe them to have any relevant impact on the primary outcome with three of them not even showing any variation within the examined cohort. As illustrated in Fig. 21. In the SCD/ECD algorithm creatinine level, hypertension, and death by stroke are only relevant within the short period between 50 and 59 years. Moreover, this algorithm disregards the combination of negative predictors It has been proven that the outcome of SCD-kidneys with high KDPI scores is comparable to ECD-kidneys with a medium KDPI35.However, the KDPI might provide certain advantages compared to the SCD/ECD labeling  and death-censored graft failure (p = 0.01) when selecting only ECD-kidneys for analogous multivariable analyses (n = 274). Therefore, we can state at least, that the KDPI provides valid and differentiated predictive information, where the ECD-criterion is just equivalently true.Another advantage of the KDPI is that it absorbs donor characteristics that are at risk of becoming (according to our data) unjustifiably important when considered separately.However, a disadvantage of the KDPI might be that it could generate a labeling effect a fortiori. Whereas a simply ECD-labeled kidney was considered to be generally usable for selected and consented patients, a high KDPI is supposed to be more discouraging. Certainly, further prospective studies have to evaluate this issue.Actually, within the Eurotransplant region the SCD/ECD rating would have no applicability as it would interfere with the Eurotransplant Senior Programm. In contrast the KDPI is applicable in all allocation subsystems. However, the application of the KDPI should be evaluated in a prospective study. Our retrospective approach can only be hypothesis generating.39, although one of the latest reviews describes this effect to be only “slight”40.Our finding of an additional adverse effect of the recipient’s BMI is supported by further studiesWe have to point out that a potential impact of the underlying renal disease, differences in the immunosuppressive therapy, infectious complications as well as anatomically, traumatically and procedurally caused graft anomalies were not considered within this study.Furthermore, this study does not contain information about the kidneys that were discarded during the follow-up period, especially information regarding their KDPI and age, the reason for discarding them and the outcome of their potential recipients. Thus, further studies need to be conducted to provide an in-depth clarification about the fate of these kidneys.The data collected for this study were analyzed retrospectively, and all inferential statistics were interpreted in an exploratory manner. It should be noted that potential errors might have been introduced, as they are inherent when maintaining a single-center database.From our data, we conclude that the KDPI can be applied to approximately predict the short-and long-term functional outcomes of RTx in a European cohort.Furthermore, our results suggest focussing on donor age as the most significant input parameter of the KDPI, as it can substantially determine the KDPI under German conditions. Presumably, the general predictive power of age is barely lower than that of the KDPI but age may serve as a simple rule of thumb to estimate the graft’s prognosis. In difficult cases it may be reasonable to calculate the KDPI and include additional information such as the patients’ BMI and the HLA-matching.Propectively, we suggest establishing an adjusted KDPI score specific to European cohorts. This score should assist transplant physicians and paitients in deciding whether to accept or decline an organ offer.Supplementary Information


Objectives: The importance of immunoglobulin G (IgG) oligoclonal bands (OCB) in the diagnosis of multiple sclerosis (MS) was reaffirmed again in the recently revised MS diagnostic criteria. Since OCB testing is based on non-quantitative techniques and demands considerable methodological experience, measurement of CSF immunoglobulin free light chains (FLC) has been suggested as quantitative alternative to OCB. We aimed to establish reference values for FLC measures and evaluate their diagnostic accuracy with regard to the diagnosis of MS.Methods: Immunoglobulin kappa (KFLC) and lambda (LFLC) free light chains were prospectively measured by nephelometry in CSF and serum sample pairs in 1,224 patients. The analyzed cohort included patients with MS, other autoimmune or infectious inflammatory diseases of the nervous system as well as 989 patients without signs for nervous system inflammation.Results: Regarding diagnosis of MS, the diagnostic sensitivity and specificity of intrathecal KFLC ratio were 93.3 and 93.7% using the CSF-serum albumin ratio-dependent reference values, 92.0 and 95.9% for intrathecal KFLC ratio applying the ROC-curve determined cut-off levels, 62.7 and 98.3% for IgG index, 64.0 and 98.8% for intrathecal IgG synthesis according to Reiber diagrams, and 94.7 and 93.3% for OCB. Diagnostic sensitivity and specificity of intrathecal LFLC were clearly lower than KFLC.Conclusions: Intrathecal KFLC and OCB showed the highest diagnostic sensitivities for MS. However, specificity was slightly lower compared to other quantitative IgG parameters. Consequently, CSF FLC may not replace OCB, but it may support diagnosis in MS as a quantitative parameter. Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS) affecting predominantly young adults and leading to neurological disability –3. CSF iSeveral studies have indicated that elevated immunoglobulin kappa (KFLC) and lambda (LFLC) free light chains in the CSF may represent a quantitative tool to demonstrate intrathecal IgG synthesis and thereby support the diagnosis of MS –28, someThe aim of the present study was (i) to prospectively measure FLC (both KFLC and LFLC) in CSF and serum by nephelometry in a large cohort, (ii) to establish reference values for FLC as a function of the blood-CSF barrier function based on patients without any clinical and laboratory signs for nervous system inflammation, and (iii) to compare the diagnostic value of different previously proposed methods to calculate intrathecal FLC synthesis, e.g., CSF-serum ratio of FLC (Q FLC), FLC index (Q FLC/Q Albumin), CSF KFLC-LFLC ratio, with well-proven indicators of intrathecal IgG synthesis (OCB and IgG Index) within the same cohort.Cross-sectional data of CSF and serum sample pairs of 1,224 patients from the Department of Neurology, University of Ulm (Germany) were investigated prospectively over a period of 18 months.The analyzed cohort included 75 patients with multiple sclerosis (MS), diagnosed according to the modified McDonald criteria , five wiOCB were detected by isoelectric focusing (IEF) on polyacrylamide gels followed by immunoblotting using an IgG-specific antibody staining. Paired CSF and serum samples were adjusted for IgG concentrations and analyzed on the same gel run as we previously described in detail . OCB werImmunoglobulin kappa and lambda free light chains (KFLC and LFLC) were measured by nephelometry (Siemens N Latex FLC kappa and lambda assays on Siemens BN ProSpec®) according to the instructions supplied by the manufacturer. The detection antibodies in these assays are monoclonal. All samples were analyzed within the same working day together with other CSF measurements according to the sampling protocol of BioMS .We calculated the CSF-serum ratio of albumin (Q Alb), KFLC (Q KFLC), and LFLC (Q LFLC) and determined the KFLC index (Q KFLC/Q Alb) and LFLC index (Q KFLC/Q Alb) by correcting for Q Alb. In addition, the CSF KFLC to LFLC ratio (CSF KFLC/CSF LFLC) was calculated.CSF leukocyte count (cells/μl), CSF total protein (g/L), CSF lactate (mmol/L), the albumin CSF-serum concentration ratio (Q Alb), CSF and serum immunoglobulin G, A, and M levels were obtained as previously described , 36.MRI scans of the brain and spinal cord were performed on a 1.5 tesla whole-body MRI  according to a previously fixed protocol including T1-weighted spin-echo (SE) axial slices with and without application of gadolinium-DTPA as well as T2-weighted SE axial slices.P-values below 0.05 were considered to be significant.Statistical analysis and the graphical representation of the data was performed using SPSS , GraphPad Prism , and R software (version 3.4.0). Differences of FLC levels between two disease groups were analyzed using the non-parametric Mann-Whitney U-test due to skewed data. The correlation between two parameters was analyzed using Spearman rank order correlation. FLC reference values dependent on Q Alb were estimated by linear quantile regression , 38 usinSensitivity was calculated as [true-positive/[true-positive + false-negative]], specificity was calculated as [true-negative/[true-negative + false-positive]]. The positive predictive value (PPV) was calculated as [true-positive/[true-positive + false-positive]], and the negative predictive value (NPV) as [true-negative/[true-negative + false-negative]]. For all diagnostic values the exact 95% confidence intervals were given .A total of 1,224 CSF-serum pairs from 1,224 patients were included in this study. Demographic, clinical and main CSF characteristics are summarized in n = 989), CSF FLC levels correlated significantly with serum FLC levels  as well as with the respective Q Alb  (In NIND (= 0.653) .To establish blood-CSF barrier related reference values for Q FLC, the Q FLC of all NIND was plotted against the respective Q Alb on the log scale. The 99% quantile estimated by linear quantile regression was indicated as the upper reference value of the respective Q FLC . This rep < 0.0001) and Q IgG according to Reiber (p < 0.0001).In the 75 MS patients, Q KFLC was positive  but showed otherwise no significant difference as compared with NIND.Patients with MS showed significantly elevated CSF KFLC levels as compared with all other groups except for CIS and CSF LFLC levels were also elevated significantly as compared with NIND, PP-PNP, P-CNS-I, and CIS . CSF KFLROC analysis was performed to determine optimal cut-off levels of Q KFLC, Q LFLC, KFLC index, LFLC index, and CSF KFLC to LFLC ratio . To distSensitivity, specificity, positive, and negative predictive values of all parameters investigated are given in Several findings support the role of B cells and intrathecal immunoglobulins in the pathogenesis of MS including (i) presence of B-cell infiltrates in the CNS parenchyma and meningeal tissues , 42, ii, 44, ii, and iv, 47 as wThe presence of OCB in the CSF is known since the 1970s , 50 as tIn line with previous studies from others and us , 18, 26 A significant correlation of CSF FLC levels (both KFLC and LFLC) with serum FLC levels and CSF-serum ratio of albumin (Q Alb) in NIND patients could be shown. Based on the large cohort of patients without any inflammatory CNS reaction we were able to introduce reference values of Q FLC (for both KFLC and LFLC) in relation to a wide range of Q Alb, which is a widely accepted quantitative measure of blood-CSF barrier function . FurtherThis study allowed to assess the diagnostic utility of the different FLC measures within one large cohort. Q KFLC  showed the highest sensitivity and the KFLC index showed the highest combined sensitivity and specificity. With regard to the diagnostic accuracy of Q KFLC in MS, however, applying the Q Alb-dependent reference values . Intrathecal KFLC and OCB showed nearly the same value (93.3 and 94.67%) with regard to diagnostic sensitivity in patients with MS, which is in line with earlier reports concerning diagnostic sensitivity of OCB in MS –58. As iDoes the determination of FLC have any advantage over the established markers of intrathecal IgG synthesis? In comparison to OCB, measurement of KFLC is reliable, rapid, methodologically simple, can be performed using either ELISA or nephelometry, and can be applied in the clinical setting together with testing of basic CSF variables. In comparison to other quantitative parameters of intrathecal IgG synthesis (according to Reiber or IgG index), Q KFLC is more sensitive.On the other hand, quantitative FLC values do not provide any insight into the clonality of intrathecal IgG, while the qualitative detection of IgG by immunoblotting can discriminate between monoclonal, oligoclonal, and polyclonal patterns.In conclusion, CSF FLC may not replace OCB but may be supportive quantitative parameters in particular cases, for example in OCB negative MS cases or equivocal OCB findings.Furthermore, this study again underlines the utility and accuracy of CSF examination in the diagnostic procedure of MS since very high diagnostic accuracy could be confirmed once again for well-established  and demonstrated for promising  markers of intrathecal IgG production.The datasets generated for this study are available on request to the corresponding author.Written informed consent was obtained from all patients in accordance with the Declaration of Helsinki, and the study was approved by the ethics committee of the University of Ulm (No. 10/20).MS, FM-Y, and HT were involved in the conception and design of the study. Data were acquired by FM-Y, MS, FB, JL, AL, MO, and HT. MS, FM-Y, UB, and HT were involved in the statistical methods and analysis. The first draft of the manuscript was designed by MS and HT, followed by critical revision of all authors. The final version for submission was approved by all authors.MS has received honoraria for speaking and/or travel from Bayer, Biogen, Sanofi Genzyme, and TEVA and research funding from the Hertha-Nathorff-Program and University of Ulm, none related to this study. JL has received honoraria for speaking and travel grants from Bayer, TEVA, CHDI, and the Movement Disorders Society. HT received funding for research projects, lectures, and travel from Bayer, Biogen, Genzyme, Fresenius, Merck, Mylan, Novartis, Roche, Siemens Health Diagnostics, Teva, and received research support from Hertie-Stiftung, DMSG, BMBF, University of Ulm and Landesstiftung BW. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


Taenia solium is known to cause human cysticercosis while T. saginata does not. Comparative in vitro and in vivo studies on the oncosphere and the postoncospheral (PO) forms of T. solium and T. saginata may help to elucidate why cysticercosis can occur from one and not the other. The aim of this study was to use in vitro culture assays and in vivo models to study the differences in the development of the T. solium and T. saginata oncosphere. Furthermore, this study aimed to evaluate the expression of cytokines and metalloproteinases (MMPs) in human peripheral blood mononuclear cells (PBMCs), which were stimulated by these oncospheres and PO antigens. T. solium and T. saginata activated oncospheres (AO) were cultured in INT-407 and HCT-8 intestinal cells for 180 days. The T. solium began to die while the T. saginata grew for 180 days and developed to cysticerci in INT-407 cells. Rats were inoculated intracranially with AO and PO forms of either T. saginata or T. solium. Rats infected with T. solium AO and PO forms developed neurocysticercosis (NCC), while those infected with the T. saginata did not. Human PMBCs were stimulated with antigens of AO and PO forms of both species, and the production of cytokines and metalloproteinases (MMPs) was measured. The T. solium AO antigen stimulated a higher production of IL-4, IL-5, IL-13, IFN-γ, and IL-2 cytokines compared to T. saginata AO. In the PO form, the T. saginata PO antigen increased the production of IL-4, IL-5, IL-13, IFN-γ, IL-1β, IL-6, IL-10, TNF-α and IL-12 cytokines compared to T. solium, suggesting that this global immune response stimulated by different forms could permit survival or destruction of the parasite depending of their life-cycle stage. Regarding MMPs, T. solium AO antigen stimulated a higher production of MMP-9 compared to T. saginata AO antigen, which may be responsible for altering the permeability of intestinal cells and facilitating breakdown of the blood-brain barrier during the process of invasion of host tissue. Taenia solium and Taenia saginata are two parasites that cause the tissue infection cysticercosis in their intermediate hosts, pigs and cows, respectively. One major difference between them is that T. solium can also cause neurocysticercosis in the human brain, while T. saginata cannot. Neurocysticercosis is thought to be the major cause of adult-onset seizures in developing countries. It is not well understood why only T. solium can survive in human tissue; however, the host inflammatory response likely plays an important role. The authors found that human immune cells stimulated with T. solium in the early stages of the parasite life cycle produced a more robust cytokine response than T. saginata. However, in the mature stage, which occurs once T. solium reaches the brain, T. solium antigens stimulated a lower inflammatory response compared to T. saginata, suggesting the parasite is able to manipulate the host immune response in some way to evade destruction. These findings may support the differences in growth observed by the authors when rat brains were inoculated with either parasite species. This study provides new insights into the different ways T. solium and T. saginata activate the immune response to survive and develop within the host. Taenia solium and T. saginata are two taeniid cestodes that cause the diseases taeniasis and cysticercosis [T. solium and T. saginata infection leading to taeniasis. In the case of T. solium, humans can also act as accidental intermediate hosts causing human cysticercosis [T. solium causes human cysticercosis, while T. saginata does not [cercosis . These acercosis . Howeverdoes not .When cysticercosis involves the central nervous system in humans, it is called neurocysticercosis (NCC). NCC is common throughout Latin America, sub-Saharan Africa, most of Asia, and parts of Oceania. Human NCC is believed to be the leading cause of acquired epilepsy worldwide ,5.T. solium and T. saginata contain a six-hooked larva called the oncosphere [T. solium activated oncosphere can then penetrate the intestinal wall. Once in the tissue, usually in the muscle or the central nervous system, the oncosphere can transform into a postoncospheral form, and completely develop into cysticerci—a larval stage that consists of a fluid-filled sac containing an invaginated scolex. When this happens, the parasite produces a variety of molecules, which modulate the host immune response in order to evade parasite destruction [The eggs of cosphere . When thtruction .T. solium and T. saginata can be obtained in vitro by co-culture of oncospheres with a monolayer of mammalian feeder cells [T. solium oncosphere and in-vitro generated PO forms can develop into cysticercus in rats causing NCC [in vitro development of the oncosphere to PO form of T. solium and T. saginata, specifically the immunological events that occur at the host/parasite interface. Also, it is not known if the development of the T. saginata oncosphere to PO form could cause NCC in the rat model as T. solium does. In vitro and in vivo models could lead to a better understanding of host-parasite relationships.The postoncospheral (PO) form is an intermediate stage between an oncosphere and a fully developed cysticercus in tissue . The PO er cells ,10. T. ssing NCC ,11. HoweHost immune cells such as macrophages, lymphocytes, and polymorphonuclear leukocytes can produce cytokines and metalloproteinases (MMPs) in order to prevent the development of the parasite ,13. BecaT. solium and T. saginata are limited. This study focused on the in vitro development of the oncosphere to the PO form for T. solium and T. saginata. These in vitro-developed larvae were then tested for infectivity in rats. Later, we obtained antigens from the T. solium and T. saginata oncosphere and PO forms to stimulate the production of cytokines and MMPs in healthy human peripheral blood mononuclear cells (PBMCs).Comparative studies on the oncosphere and the PO form of T. solium and T. saginata PO forms. Cells were incubated at 37°C in 5% CO2 and grown in a specific medium as recommended by ATCC . The medium was changed every two days. Once cell confluency was obtained, cells were harvested using trypsin-EDTA . Cells were placed into 24-well plates (1x105 cells per well) for maturation assay. The assays described below were performed when cells formed a monolayer.HCT-8 and INT-407 cells, obtained from the American Tissue Culture Collection , were used to obtain et al [et al [T. solium) or 90 minutes (in the case of T. saginata) with artificial intestinal fluid , 200 mg Na2CO3, and 1 ml of fresh porcine bile (for T. solium), or 1 ml of fresh bovine bile (for T. saginata), with enough RPMI 1640 medium (pH 8.04) to make 100mL). After activation, the oncospheres were washed three times with RPMI medium and counted using a Neubauer chamber.Tapeworms were collected after medical treatment of newly diagnosed patients, as described by Jeri et al . Hatchinl [et al . The eggin vitro maturation assays with INT-407 and HCT-8 monolayer cells, using T. solium and T. saginata activated oncospheres, were conducted in order to compare the morphological characteristics during development of each species using the methodology reported by Chile et al [Parallel le et al . Ten thoT. saginata AO and PO forms can develop into viable cysts in vivo, 15-day old Holtzman rats, purchased from Universidad Peruana Cayetano Heredia, Lima, Peru, were infected intracranially (in the bregma) with oncospheres and 15-day old PO forms from either species following the methodology reported by Verastegui et al., 2015 [To determine if l., 2015 . Rats weT. solium AO in 100 μL of saline solution; seven rats were inoculated with 180 T. saginata AO in 100 μL of saline solution. The negative control was 2 rats inoculated with saline solution.Six rats were inoculated with 180 T. solium 15-day PO forms in 100 μL of saline solution, eight rats were inoculated with ten T. saginata 15-day PO forms in 100 μL of saline solution, and five rats were inoculated with 100 μL of saline solution as a control. A 24-gauge syringe needle was used.Eight rats were inoculated with ten After four months, necropsy was performed. Rats were anaesthetized with ketamine (100 mg/kg body weight) and xylazine (5 mg/kg body weight). Anaesthetized rats were perfused with 200 ml of PBS and then with 100 ml of 4% paraformaldehyde in PBS. Brains were carefully removed, post-fixed for 24 hours at 4°C with 4% paraformaldehyde in PBS, and stored in 70% ethanol. Brains were observed macroscopically to identify extraparenchymal cysticerci. Five millimeter coronal brain sections were cut until the intraparenchymal cysticerci were observed.Antigens were obtained from AO and 30 day-PO forms of both parasites. AO and PO were obtained as described above. Parasites were rinsed three times with PBS buffer, sonicated, and centrifuged at 10,000g for 15 min at 4°C. The supernatant  was separated, and proteins were quantified using Bradford Protein Assay (Bio-Rad) and stored at -70°C until ready for use.TM PLUS, GE Healthcare) for 10 minutes at room temperature for the separation of mononuclear cells. Cells were again suspended in RPMI medium plus 5% of inactivated human serum. Cell viability was evaluated with trypan blue and counted in a Neubauer chamber. Each well contained 2x105 group of cells was cultured in a 96-well plate at 37°C with 5% CO2 for 48 hours in an RPMI medium containing 5% of inactivated human serum. Cells were stimulated with 5 μg/ml of phytohemaglutinin (PHA) as the positive control, 20 μg/ml of AO antigens , and 20 μg/ml of antigens from PO forms at 30 days of maturation . At the end of the incubation period, PBMCs were harvested and centrifuged for 10 min at 400g, and supernatants were collected and stored at −70°C until tested for cytokine and metalloproteinase content by multiplex analysis.Healthy volunteers (n = 13) with negative serology for NCC were invited to participate in this study. After the volunteers signed an informed consent form, 10 mL of venous blood was collected from each non-infected donor. PBMCs were collected from 10 ml EDTA blood. The blood samples were centrifuged at 400g on a Ficoll-Hypaque gradient (Ficoll–Paque MILLIPLEX MAP kit High Sensitivity Human Cytokine Magnetic Bead Panel (Millipore) was used to measure cytokines , and Fluorokine MAP  was used to measure MMP (MMP-2 and MMP-9) in the supernatant of stimulated PBMCs following the manufacturer’s instructions. The cytokines and MMP were detected by the Bio-plex 200 system  using Luminex xMAP technology.Experiments were done according to the Guide for care and Use of Laboratory Animals of the National Institutes of Health. The protocol was approved by the Committee on the Ethics Animals of the Universidad Peruana Cayetano Heredia, Lima, Peru (Permit Numbers: 61242).For cytokines and MMP assays, the median fluorescent intensities of the beads with the cytokines or MMP now bound were converted to concentrations (pg/ml), using a five-parameter logistic model using the Bio-Plex Manager™ 6.0 software . Each MMP´s or cytokine’s concentration was normalized by subtracting the data of medium alone (control). The Mann Whitney test was used to compare two unpaired groups, and ANOVA followed by Tukey’s post-test was used for three or more group using the Prism V 6.0 statistical program (GraphPad). A P-value of <0.05 was considered statistically significant.T. saginata and T. solium developed from the oncosphere to the PO form in both cell lines, INT-407 and HCT-8 cells  species, both AO forms . Neverth AO form  compared PO form .T. solium and T. saginata forms. The T. solium AO stimulated higher MMP-9 production compared to the T. saginata AO, while in PO form there was no significant difference between both species  response, the predominant protective immune response against helminthic infections [H1) response, which is thought to be responsible for destruction of the parasite [H2 response is important in protecting against extracellular helminthic parasites through suppression of the TH1 response, neutralization of toxins, and defense of the host against damage [We observed that fections . On the parasite . The TH2t damage , 24.H1 and TH2 type responses. In the case of the T. solium oncosphere, it appears a mix immune reaction of TH1/ TH2 type response that aids survival of the parasite. Perhaps in doing so, T. solium oncospheres are able to migrate from the vasculature to the brain, where they develop into the PO form. Similar mixed TH1/TH2 phenotypes have been observed in patients with NCC [H2-type response is associated with asymptomatic disease [Inflammatory reactions are dependent on a delicate balance between Twith NCC , 13 wher disease , 26.In both species, the 30-day PO form generated an overall greater inflammatory response than the AO form. Levels cytokines increased when exposed to equal concentrations of PO vs AO antigen, suggesting that antigen composition changes as the parasite is maturing .T. saginata 30-day PO form stimulated a profile of pro-inflammatory cytokines , plus a mix of TH1 and TH2 related cytokines  that was stronger than the response produced by the T. solium 30-day PO form. IL-6, a pro-inflammatory cytokine, plays a role in the death of microorganisms by stimulating the behavior of neutrophils [T. saginata PO form and prevents development of the cyst in vivo. Although these cytokines do have the suggested properties mentioned, they also could be involved in complex networks with mixed effects with respect to inflammation, for example IL-6 has been shown to exhibit anti-inflammatory properties [However, the trophils ; and TNFtrophils . Togetheoperties .T. solium oncospheres stimulated increased MMP-9 production in PBMCs compared to production by T. saginata oncospheres. MMP-9 is an endopeptidase produced by neutrophils, macrophages, monocytes, and intestinal epithelial cells [T. solium stimulates the production of MMP-9 as a means of enhancing epithelial permeability in order to pass restrictive biological barriers like the intestine and the BBB during early stages of infection.al cells ,31. It cal cells ,32,33. Mal cells ,35 and ial cells . We hypoT. saginata PO forms are capable of developing into cysticerci in the human cell line INT-407 while not in HCT-8 cells. In vivo, T. saginata fails to develop into cysticerci in the rat brain, suggesting there are factors in the host immune system (that are not present in the in vitro culture) that destroy the parasite. In the oncosphere stage, T. solium stimulated a strong mix of TH1 and TH2-related cytokines and MMP-9 production in healthy PBMCs, which may mediate the inflammatory response and promote oncosphere survival in the vasculature, aiding the entrance of oncospheres into the brain. In the PO form, T. saginata stimulated a strong pro-inflammatory and mix of TH1/TH2-related cytokines, responses that could be causing destruction of the parasite in the tissue. These differences between both species of Taenia found in vitro and in vivo could explain why the larval stage of T. saginata does not develop in the human host, while T. solium does, despite having similar life cycles.In conclusion, we have found novel evidence to suggest that S1 FigTaenia solium antigen B) Taenia saginata antigen.A) (TIF)Click here for additional data file.S2 Fig(TIF)Click here for additional data file.


Staphylococcus epidermidis cerebrospinal fluid (CSF) shunt infection is a common complication of hydrocephalus treatment, creating grave neurological consequences for patients, especially when diagnosis is delayed. The current method of diagnosis relies on microbiological culture; however, awaiting culture results may cause treatment delays, or culture may fail to identify infection altogether, so newer methods are needed. Staphylococcus epidermidis cerebrospinal fluid (CSF) shunt infection is a common complication of hydrocephalus treatment, creating grave neurological consequences for patients, especially when diagnosis is delayed. The current method of diagnosis relies on microbiological culture; however, awaiting culture results may cause treatment delays, or culture may fail to identify infection altogether, so newer methods are needed. To investigate potential CSF biomarkers of S. epidermidis shunt infection, we developed a rat model allowing for serial CSF sampling. We found elevated levels of interleukin-10 (IL-10), IL-1β, chemokine ligand 2 (CCL2), and CCL3 in the CSF of animals implanted with S. epidermidis-infected catheters compared to sterile controls at day 1 postinfection. Along with increased chemokine and cytokine expression early in infection, neutrophil influx was significantly increased in the CSF of animals with infected catheters, suggesting that coupling leukocyte counts with inflammatory mediators may differentiate infection from sterile inflammation. Mass spectrometry analysis revealed that the CSF proteome in sterile animals was similar to that in infected animals at day 1; however, by day 5 postinfection, there was an increase in the number of differently expressed proteins in the CSF of infected compared to sterile groups. The expansion of the proteome at day 5 postinfection was interesting, as bacterial burdens began to decline by this point, yet the CSF proteome data indicated that the host response remained active, especially with regard to the complement cascade. Collectively, these results provide potential biomarkers to distinguish S. epidermidis infection from sterile postoperative inflammation. Staphylococcus epidermidis being the leading species  shunt placement is the most common treatment for hydrocephalus, with up to 33,000 CSF shunts placed each year . Unfortu2– species . Therefo species .Currently, the diagnosis of shunt infection relies on isolating a pathogen from CSF culture; however, there are numerous challenges with this methodology , 7. FirsIn the case of a negative CSF culture, clinicians must rely on serum laboratory results, other CSF indices , and clinical presentation to determine if an infection is present . However12–3CL1), chemokine ligand 2 (CCL2), and CCL3  can be used to identify proteins related to the host immune response and/or pathogen virulence factors that may prove useful from a diagnostic perspective. MS has been widely used to identify biomarker candidates in several neurodegenerative disorders, such as multiple sclerosis, Parkinson’s disease, and Alzheimer’s disease –22. Pert–fections . To expaS. epidermidis catheter infection that supports serial CSF sampling, to identify potential biomarkers of infection in the CSF that is routinely collected as part of clinical care in patients with suspected shunt infection. CSF samples from animals with S. epidermidis-infected catheters revealed elevated IL-10, IL-1β, CCL2, and CCL3 levels compared to sterile controls. Infected animals also displayed increased CSF neutrophil infiltrates at day 1 postinfection compared to controls, with a shift to predominately monocytes/macrophages at day 5 postinfection. MS analysis revealed an increased number of proteins in the CSF of infected animals, particularly related to the complement pathway. Interestingly, elevated expression levels of several proteins in the CSF persisted in the face of declining infectious burdens. These results have identified several potential biomarkers for further study in animal and human trials of S. epidermidis shunt infection.Here we utilized a rat model of S. epidermidis 1457 bacteria. This approach has been widely used in mouse models of infection in the CSF and periphery (P = 0.001) (P = 0.149)  , which i= 0.001) . By day = 0.149) . QuantifS. epidermidis catheter infection, CSF was analyzed for the presence of IL-1β, IL-6, IL-10, CCL2, CCL3, chemokine (C-X-C motif) ligand 1 (CXCL1), TNF-α, VEGF, and CX3CL1 by a multianalyte microbead array. Brain homogenates from tissues immediately surrounding the catheter tract were included for comparison. The CSF of animals with S. epidermidis-infected implanted catheters had significantly higher levels of IL-10 (P = 0.005), IL-1β (P = 0.002), CCL2 (P = <0.001), and CCL3 (P = <0.001) at day 1 postinfection than those of animals receiving sterile catheters (S. epidermidis-infected catheters had higher levels of IL-10 (P < 0.001), IL-1β (P < 0.001), CCL2 (P < 0.001), and CCL3 (P < 0.001) at day 1 postinfection, demonstrating that the CSF inflammatory milieu mirrors that of the brain tissue during early infection. As bacterial burdens decreased at day 5 postinfection, levels of IL-1β (P < 0.001), CCL2 (P = 0.010), and CCL3 (P < 0.001) remained significantly elevated in the brain tissue of animals with S. epidermidis-infected catheters  (P = 0.054) or brain tissue (P = 0.683) at day 5 postinfection (3CL1 in CSF or brain tissue at either time point (data not shown).To assess the utility of CSF cytokines and chemokines as biomarkers for atheters . Likewisatheters ; however= 0.451) . No signnfection . In addiS. epidermidis infection, we next examined whether this translated to changes in leukocyte influx. Neutrophil infiltrates (CD45+ CD11b+ RP1+) were significantly increased in the CSF of animals implanted with S. epidermidis-infected versus sterile catheters (P = 0.001) (+ CD11b+ RP1−) in the CSF of animals receiving infected versus sterile catheters at either time point (Since the levels of numerous chemokines were elevated in the CSF during = 0.001) , consist= 0.001) . There wme point . The pauS. epidermidis-infected catheters  revealed distinctions between the CSF proteomes of sterile and infected animals at both days 1 and 5 postimplantation . Heat maatheters . At day atheters .MS data were also evaluated using differential expression analysis. At day 1, a total of 39 proteins were differentially expressed between sterile and infected groups, including several components of the complement cascade that were significantly enriched during infection . The numS. epidermidis catheter infection . At day 1 postinfection, a total of 6 biological processes were enriched, with 2 to 7 proteins present in each category. At day 5 postinfection, the number of biological processes increased to 13, even in the context of reduced bacterial burdens. When comparing pathways that were common between the two time points, the number of proteins at day 5 postinfection was increased, suggesting progressive pathology.Pathway analysis of the differentially expressed proteins identified several biological processes that were active during P31720 [P = 0.005] and P31721 [P = 0.024]) (P = 0.003), complement C8 chains (UniProt accession numbers D3ZWD6 [P = 0.048] and D3ZPI8 [P = 0.004]), complement factor properdin (P = 0.005), and complement C3 (UniProt accession number P01026 [P = 0.009]) , complement C1r (P < 0.001 and P = 0.006), complement C3 (UniProt accession number MORBF1 [P = 0.010 and P = 0.009]), and complement factor B fragment (P = 0.031 and P = 0.011) were significantly higher in the infected group at both days 1 and 5 . Complement proteins have been found to play significant roles in synaptic pruning during CNS development as well as in a variety of neurological disorders, such as Alzheimer’s disease and multiple sclerosis –43. Give–S. epidermidis is the most common cause of CSF shunt infections, other bacteria are capable of causing disease. Our previous pilot study revealed that in human CSF, there were differences in CSF inflammatory mediator concentrations in Gram-positive versus Gram-negative infections, with higher levels of the proinflammatory cytokines IL-1β, CX3CL1, CCL2, and CCL3 seen in response to Gram-negative shunt infection . The protocol for animal use was approved by the University of Nebraska Medical Center Institutional Animal Care and Use Committee (protocol number 16-091-09-FC) and is compliant with National Institutes of Health guidelines for the use of rodents. Animals were housed 2 to 3 per cage with species-appropriate enrichment in a 12-h light-dark cycle. Food and water were available S. epidermidis 1457 was kindly provided by Paul Fey . This isolate was recovered from a central venous catheter and characterized by Mack et al. and was previously used in a murine model of central nervous system catheter infection  were incubated overnight with catheter . Rats weAt days 1 and 5 after catheter insertion, rats were anesthetized with an i.p. injection of ketamine and xylazine , the back of the neck was shaved, and animals were placed in a stereotactic apparatus. Once positioned, the back of the neck was swabbed with betadine, and a percutaneous cisterna magna puncture was performed using a 25-gauge winged infusion set  with a guard to leave 4 mm of the needle tip exposed . A totalCatheters and brain tissue were collected at days 1 and 5 postinfection as previously described , 26. Bri3CL1). Results were analyzed using Multiplex Assay Analysis software and normalized to the sample volume.Levels of inflammatory mediators in the CSF and brain tissue were measured with a rat microbead array system according to the manufacturer’s instructions . This assay allowed for simultaneous measurement of 9 different cytokines and chemokines in a single 50-μl CSF or brain supernatant sample, including IL-1β, IL-6, IL-10, CCL2, CCL3, chemokine (C-X-C motif) ligand 1 (CXCL1), TNF-α, vascular endothelial growth factor (VEGF), and fractalkine  analysis based on the expression of characteristic cell markers: macrophages/monocytes (CD45+ CD11b+ RP1−) and neutrophils (CD45+ CD11b+ RP1+) (To characterize CSF leukocytes associated with b+ RP1+) , 48.For serial collection of CSF at days 1 and 5 postinfection, rats were anesthetized with inhaled isoflurane (1% to 5% to effect). The back of the neck was shaved, and animals were placed in a stereotactic apparatus. Once positioned, the back of the neck was swabbed with betadine, and a percutaneous cisterna magna puncture was performed using a 25-gauge winged infusion set with a guard to leave 4 mm of the needle tip exposed  . A total4HCO3) for 1 h at 37°C and dehydrated with ACN. Reduced proteins were alkylated with 50 mM iodoacetamide (IAA)–50 mM AmBic for 20 min in the dark, with rotation. Gel pieces were then dehydrated for a second time with ACN to remove all reagents. Mass-spectrometry-grade trypsin (15 ng/μl) (Promega) was added to the samples and incubated for 30 min while on ice. After removal of excess trypsin, 25 mM AmBic was added to cover the gel pieces, which were incubated overnight at 37°C. Digested peptides were then extracted from the gel with a 50% ACN–0.1% trifluoroacetic acid solution. Samples were dried in a SpeedVac, dissolved in 15 μl formic acid (FA) (0.1%), and submitted for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis.CSF samples were processed by electrophoretic protein separation. Gels were then stained with Coomassie brilliant blue G-250 dye (Thermo Fisher) for 2 h and left to destain overnight in destaining solution (10% acetic acid–20% methanol). The destained gel pieces were incised, washed with high-performance liquid chromatography (HPLC)-grade water, and dehydrated with neat acetonitrile (ACN). Proteins were then reduced with 2 mM Tris(2-carboxyethyl)phosphine (TCEP) in 50 mM ammonium bicarbonate (AmBic) , activation type of higher-energy collisional dissociation (HCD), HCD collision energy of 40%, and first mass of m/z 110. Stainless steel emitters were purchased from Thermo Fisher .In-gel-digested peptide samples were analyzed using an Orbitrap Fusion Lumos instrument coupled with an UltiMate 3000 HPLC system (Thermo Scientific). A total of 5 μl (500 ng) of each sample was loaded onto the trap column  using FA (0.1%) and resolved in a rapid-separation liquid chromatography (RSLC) column . Samples were eluted using a 90-min linear gradient of ACN (4% to 45%) in 0.1% FA. The parameters for all experiments were as follows: nanospray needle voltage in positive mode of 2,000 V, column flow rate of 0.3 μl/min, loading pump flow rate of 4 μl/min, and inject mode of μl PickUp. The Orbitrap scan mode was used for MS/MS, with a resolution of 120,000 and a scan range of t test with SigmaStat  at the 95% confidence interval. A P value of less than 0.05 was considered statistically significant.For cytokine and leukocyte infiltrate measurements, significant differences between experimental groups were determined using unpaired Student’s P value with 1,000 random permutations of protein expression between the two groups. To adjust the false discovery rate due to multiple testing, we applied the Benjamini-Hochberg adjustment of the P value  to this multidimensional expression profile to reduce the dimension to fewer components. We used the “pheatmap” package in R  to generate a heat map of expression for the highly variable proteins , 50. We  P value . Inbuilt P value . We used P value . Statist


We also found very high CSF levels of the pro-inflammatory interferon gamma-induced protein 10 (IP-10/CXCL10) in HIV regardless of cognition, but elevated CSF interleukin 8 (IL-8/CXCL8) only in HIV-NC but not HAND. Eleven HIV-infected subjects underwent repeat CSF collection six months later and showed strongly correlated longitudinal changes in p-Tau181 and IL-8 levels (R = 0.841). These data suggest reduced IL-8 relative to IP-10 and reduced p-Tau181 to characterize HAND.HIV-associated neurocognitive disorder (HAND) is a common condition in both developed and developing nations, but its cause is largely unknown. Previous research has inconsistently linked Alzheimer’s disease (AD), viral burden, and inflammation to the onset of HAND in HIV-infected individuals. Here we simultaneously measured cerebrospinal fluid (CSF) levels of established amyloid and tau biomarkers for AD, viral copy numbers, and six key cytokines in 41 HIV-infected individuals off combination anti-retroviral therapy (14 with HAND) who underwent detailed clinical and neuropsychological characterization, and compared their CSF patterns with those from young healthy subjects, older healthy subjects with normal cognition, and older people with AD. HAND was associated with the lowest CSF levels of phosphorylated tau (p-Tau With more than 1.1 million Americans currently living with HIV4, HAND is a more common cognitive disorder than frontotemporal dementia5 or Lewy body dementia6. In the era of combination anti-retroviral therapy (cART)7, severe HIV-associated dementia is less common in developed nations but milder forms of HAND10 can still interfere with job performance and compliance with the complex multi-drug combination11. At the same time, accurate clinical diagnosis of mild HAND (such as asymptomatic neurocognitive impairment and mild neurocognitive disorder)12 remains challenging because of common confounds . Biofluid or imaging markers have the potential of enhancing early and accurate detection of mild HAND. Such biomarkers will be especially important in drug development if they also reflect known disease mechanisms or provide new pathophysiologic insight.HIV-associated neurocognitive disorders (HAND) can affect up to 50% of people living with HIV13, no neuropathologic process has been consistently identified in HAND14. Gliosis was the most common finding16, and inclusions associated with age-related neurodegenerative disorders such as Alzheimer’s disease (AD) were variably identified. Cerebral PET imaging for neuritic plaques also failed to show increased amyloid deposition in HAND17. On the other hand, some ante-mortem cerebrospinal fluid (CSF) studies examining established AD biomarkers paradoxically showed changes in HAND similar to AD. Two US studies – but not one Dutch study – revealed reduced levels of beta-amyloid 1–42 (Aβ42) suggestive of amyloid deposition20, while levels of total tau (t-Tau) and tau phosphorylated at threonine 181 (p-Tau181) associated with HAND can be increased, normal or reduced23. Because we25 and others26 have shown that African American race and CSF proteins also influence measured CSF AD biomarker levels, an investigation into the relationship between these biomarkers and HAND must account for race and age by recruiting the appropriate comparison groups. Taking advantage of the standardized biofluid collection, handling, and analysis protocols across the HIV and Cognitive Clinics at Emory University, we analyzed AD biomarkers in a cohort of 143 subjects, including 41 infected with HIV who were off (n = 39) or naïve to cART (n = 2), 33 young healthy subjects with normal cognition (NCyoung), 44 older healthy subjects with normal cognition (NColder), and 25 older subjects with mild cognitive impairment or mild dementia due to AD (MCI/AD). We further explored whether CSF AD biomarker levels in HAND were influenced by viral load and altered levels of key CSF cytokines linked to innate immunity or T-cell-related pathways , including interferon-γ-inducible protein 10 (CXCL10/IP10), interleukin 8 (CXCL8/IL-8), fractalkine (CX3CL1), tumor necrosis factor alpha (TNF-α), macrophage-derived chemokine (MDC), and interleukin 10 (IL-10). Finally, to lend support for a mechanistic link between AD biomarkers and inflammation, we examined in a group of HIV subjects with serial CSF collection whether the two biomarker sets underwent parallel changes over time.Whereas post-mortem studies commonly associated viral infection as a key mechanism in HIV-associated dementia181) according to cognition , HIV status (infected or not-infected), and age , HIV with normal cognition (HIV-NC) and HAND were both associated with lower p-Tau181 and Aβ42 levels than NCyoung , and having HAND  were independently associated with reduced p-Tau181 levels, while older age  and cognitive impairment due to AD  were associated with increased p-Tau181 levels. For Aβ42, lower levels were associated with cognitive impairment , cognitive impairment due to AD , and female gender . Hence, reduced p-Tau181 was a feature of both HIV (regardless of cognition) and additionally HAND, while reduced Aβ42 was associated specifically with AD and non-specifically with cognitive impairment. Importantly, there was a strong correlation between Aβ42 and p-Tau181 levels in the younger population, which has not been previously reported in older healthy controls from AD studies. A review of prior studies which identified HIV/HAND-associated alterations in CSF AD biomarkers also showed four out of five previous studies to report changes in Aβ42 and tau along the same direction . HAND subjects had higher plasma viral load than HIV-NC subjects  = 5.038, p = 0.031) associated with decreased IL-8 levels  = 3.461, p = 0.071). Therefore, the selectively attenuated or muted IL-8 changes in HIV were a unique cytokine feature for HAND.CSF HIV-NC and HAND were also both associated with higher levels of multiple correlated cytokines than NC-10 Fig. , with IL30. To do so, we first converted each individual’s IL-8 and IP-10 levels into Z-scores (using NCyoung and NColder as the normative) to allow for easier comparison of two cytokines’ effect sizes according to each standard deviation alteration32. We then examined, within each individual, IL-8 and IP-10 levels relative to each other by difference between the two Z-scores (IL-8 and IP-10)33. In other word, an individual with proportional increase in IL-8 and IP-10 would have a Z-score difference of 0, while another individual with disproportional increase in IP-10 would have a negative Z-score difference (bias towards IP-10). Through this analysis, we found that HAND was associated with the greatest unopposed change in IP-10 relative to IL-8, while the other groups had relatively balanced changes in IL-8 and IP-10 , we next tested whether these two markers undergo paralleled changes over time. Follow-up CSF samples were obtained from eleven subjects  six months after the baseline collection. When change in biomarker levels were log2-transformed to give equal weight to increases and decreases, we found longitudinal changes in p-Tau181 levels to strongly correlate with changes in IL-8 , as well as increased IP-10 levels without accompanying increase in IL-8. These cross-sectional differences may point to a mechanistic link between AD biomarkers and inflammation – at least in HIV, because we also found highly correlated changes in IL-8 and p-Tau181 levels over time. Because CSF Aβ42 levels correlated directly with p-Tau181 levels in younger subjects regardless of HIV status (rather than inverse pattern in AD), we add to the growing literature that CSF biomarkers do not suggest a link between HAND and AD.Here we analyzed CSF AD biomarkers and cytokines in a group of HIV-infected and HIV-negative subjects to simultaneously examine two pathways previously implicated in HAND. We identified HAND to be characterized by reduced (but correlated) levels of two AD biomarkers , although the magnitude of change from this is likely limited39. Non-HIV infected astrocytes release IL-8 in vitro in response to HIV-infected monocyte-derived macrophages40, tat protein41, and gp12042. IL-8 increase in HIV may then reflect the number of non-infected astrocytes capable of responding to the virus. On the other hand, astrocytes genetically engineered to express HIV-1 Nef in vitro show markedly elevated IP-10 mRNA and protein levels, in keeping with the post-mortem finding of elevated astrocytic IP-10 in HIV-infected brains43. Therefore, the balance between CSF IL-8 and IP-10 levels potentially reflects the health of astrocytes in HIV-infected subjects. Because our study of HIV-infected patients off cART differs in design from prior studies involving mostly of patients on active cART, diminished CSF IL-8 levels in HAND may be reversible with treatment even though this was not universally observed in our small cohort of longitudinally characterized patients. Alternatively, discrepant CSF IL-8 findings in HAND may result from dual infection (more common HAND)44 or race-associated differences in IL8 polymorphism46. A larger longitudinal CSF study or a quantitative post-mortem analysis of HIV-infected vs. non-infected astrocytes will be necessary to fully test this hypothesis.CSF IL-8in vitro evidence of HIV Tat protein47 or peptide48 inhibiting the primary Aβ42-degrading enzyme neprilysin, and HIV Gag protein mediating the release of Aβ fragments from the amyloid precursor protein49. We have ourselves found in vitro evidence of mixed neprilysin inhibition by HIV Tat according to enzyme concentration, and acknowledge the potential in vitro overlap between HIV proteins and cellular processes capable of clearing and generating AD-related Aβ peptides (unpublished data). However, most studies now (including ours)29 have shown decreases in Aβ42 and tau markers, a pattern quite distinct from the AD profile of decreased Aβ42 but increased tau markers. Human studies therefore strongly favor reduced Aβ42 in HAND as a result of younger age instead of AD. While we do not rule out the possibility of cerebral amyloid deposition through non-AD related mechanisms in HAND17, the positive identification of reduced p-Tau181 levels and decreased IL-8 (relative to IP-10) in HAND warrant further studies on how paired immune dysregulation and tau phosphorylation may contribute to neuronal dysfunction in HIV.CSF AD proteins and cytokines have each been analyzed in HIV and HAND in previous studies Table , but oft181 levels. We and others have found aging to be associated with increasing CSF p-Tau181 levels, and it is not readily apparent why HAND would be associated with greater youthfulness in the brain. Tau hyperphosphorylation was previously observed in brains of a transgenic rat model of HIV50, and a post-mortem study found the greatest brain tau pathology in patients treated with cART. Our observed association between cognitive impairment and low p-Tau181 levels in HIV-infected patients off cART is then in keeping with inadequate viral suppression. It is important to note that while elevated t-Tau and p-Tau181 levels have been proposed as markers of general neurodegeneration and tau hyperphosphorylation in AD51, such relationships are not seen in other common degenerative disorder with rare exception. In fact, we and others have found reduced CSF p-Tau181 levels in frontotemporal lobar degeneration with lesions immunoreactive to TDP-4353, and, to a lesser degree, in Parkinson’s disease54. CSF levels of t-Tau and p-Tau181 should then not be used as surrogate markers of neurodegeneration in HIV, but HIV does represent a non-AD process that dynamically alters CSF p-Tau181 levels at a rate much faster than observed in other conditions. An improved understanding of if and how HIV infection, astrocytic health, and cART modify CSF p-Tau181 levels will likely enhance the interpretation of this marker beyond just HIV or HAND.It is not known how higher viral load or reduced IL-8 relative to IP-10 are mechanistically linked to reduced CSF p-Tau55 which leads to increased tau phosphorylation, while calcineurin – an abundant brain phosphatase – can dephosphorylate tau. Calcineurin is upregulated in vitro by neurons exposed to HIV gp12056, and is involved in reactivating latent HIV57. Calcineurin inhibition in mice genetically engineered to express gp120 also lead to reduced neuroinflammation and neuropathology58. Because of the floor effect in CSF HIV viral load measurements, p-Tau181 levels may provide greater sensitivity in monitoring the brain impact of HIV infection especially given the new automated platforms for AD biomarkers59. Future studies should incorporate measures of calcineurin-related peptides, markers of neurodegeneration  as well as p-Tau181 and cytokines to better validate this biomarker combination in HIV and HAND.Among candidate mechanisms capable of modifying tau in HIV, HIV-1 Tat enhances CDK5 translocation181 levels, a relative decrease in IL-8 compared to IP-10, and a modest increase in plasma viral load. The strong correlation in the first two markers’ longitudinal changes suggest a shared mechanism independent from plasma viral load, and the identification of fluid biomarkers may help clarify the transition between HIV-NC and HAND. These markers can potentially be used in conjunction with routine viral measures to track insidious cognitive decline, and CSF p-Tau181 levels should be tested as a surrogate biomarker of response in clinical trials targeting inflammation in HAND.In sum, we report that HAND is characterized by lower CSF p-TauThe studies were approved by the Emory University Institutional Review Board and written informed consent was obtained from all subjects. All experiments were performed in accordance with the US Common Rule (45 CFR Part 46), the Declaration of Helsinki, and Emory University policies.60. Briefly, each subject underwent standard medical evaluation and blood tests  to rule out reversible causes of cognitive impairment, followed by standardized neuropsychological testing according to the most recent HAND criteria12. For this study, participants were excluded if there was a (1) history of neurologic disease known to affect memory , (2) ongoing substance use including heavy alcohol consumption in the past 30 days (>7 drinks per week for women and >14 drinks per week for men), (3) serious mental illness , or 4) history of treated syphilis and a persistently positive rapid plasma regain titer of 1:4 or less. HIV-infected subjects were further invited to return for a 6-month follow-up visit, and 11 individuals returned for the follow-up visit and underwent a second CSF collection.HIV-infected adult subjects without subacute cognitive decline over the prior 30 days AND were off cART  were recruited at the Emory University Center for AIDS Research (CFAR) as previously described61: (1) Trailmaking Part A, (2) Trailmaking Part B, (3) Hopkins Verbal Learning Test total learning, (4) Hopkins Verbal Learning Test delayed recall, (5) Brief Visuospatial Memory Test-Revised learning, (6) Brief Visuospatial Memory Test-Revise delayed recall, (7) Grooved Pegboard (dominant), (8) Grooved Pegboard (non-dominant), (9) Finger Tapping; (10) Stroop Color Naming, (11) Stroop Color-Word interference, (12) Symbol Digit Modalities Test, (13) Letter Fluency , and (14) Category fluency . These tests were selected in order to examine at least five domains as recommended. Age, sex-, race-, and education- adjusted T-scores across 14 subtests were averaged to generate a composite neuropsychological score (NPT-14), and the validated global deficit score (GDS) was also calculated with a score ≥ 0.5 to indicate cognitive impairment62. A modified standard regression-based correction was used to account for longitudinal practice effect when determining changes in neuropsychological performance in follow-up63.All HIV-infected participants underwent comprehensive neuropsychological analysis for the assessment of HAND using 14 tests commonly used in studies of cognition and HIVyoung were also recruited at CFAR (n = 13) or as part of a broader biobanking protocol (n = 18) at Emory University. For comparative purposes, we additionally analyzed samples from older African Americans and Caucasians with normal cognition (NColder) or MCI/AD (CSF t-Tau/Aβ42 ≥ 0.39 based on a biomarker-autopsy cohort)64 recruited under a separate study25. All these subjects underwent standardized clinical examination, neuropsychological analysis, consensus diagnosis process, and MR imaging as previously described65.NC60. Plasma samples were centrifuged, aliquoted, labeled, and stored at −80 °C until analysis. CSF was collected using a modified Alzheimer’s Disease Neuroimaging Initiative protocol with minor modifications. At CFAR , CSF was obtained without overnight fasting using a 20-Gauge cutting edge needle directly into a 15 mL polypropylene tube, centrifuged to remove cellular debris (1800 RPM for 10 min), aliquoted, labeled, and immediately frozen. For other subjects, CSF was collected without overnight fasting using a 24-Gauge atraumatic needle directly into a 15 mL polypropylene tube, aliquoted, labeled, and frozen. We previously showed that CSF centrifugation to remove cellular debris did not influence measured cytokine levels, and samples collected with and without centrifugation can be analyzed together66.In all subjects, plasma and CSF samples were obtained on the same day as neuropsychological analysis10-transformed because of their non-normality.Plasma and CSF HIV RNA levels were measured in the real-time HIV-1 RT-PCR assay , with the lowest limit of HIV detection at 40 copies/mL. Plasma and CSF viral load were log181) levels were measured in a Luminex 200 platform using Alzbio3  as previously described52. Our center achieves an average inter-plate coefficient of variation (CV) of 13% for Aβ42, 10% for t-Tau, and 11% for p-Tau181. Levels of six CSF cytokines  were measured using Milliplex MAP Human Cytokine/Chemokine Magnetic Bead Panel  in a Luminex 200 platform following the manufacturer’s protocol except two 100 μL aliquots of CSF were used for duplicates. These cytokines were previously selected from a larger panel because of assay sensitivity and intermediate precision in CSF cytokine analysis. In our laboratory, we achieve average intermediate precision (in experiments performed over 9 days) of 9.4% for TNF-α, 12.9% for MDC, 14.7% for IL-7, 4.8% for IP-10, 12.0% for IL-10, 9.2% for IL-9, and 7.6% for IL-8. Freeze-thawing experiment using CSF from six separate subjects showed significant degradation over two freeze-thaw cycles for MDC and TNF-α , and we previously showed light centrifugation to have minimal impact on these CSF cytokine levels.CSF AD biomarker . ANCOVA was used to determine main effects of HIV, cognition, and the interaction term between HIV and cognition on CSF biomarker levels, including p-Tauyoung and NColder were calculated for each cytokine, and every individual’s IL-8 and IP-10 levels were transformed into Z-scores32. Z-score differences between IL-8 and IP-10 were calculated by subtracting the former’s Z-score by the latter’s Z-score33.To calculate Z-scores for IL-8 and IP-10, mean and standard deviation values from a combined cohort of NC181, and Aβ42 levels. The fold-change values then underwent log2-transformation to give equal weight to increases and decreases. Because viral load was already analyzed after log10 transformation, the change in plasma viral load was represented by the difference in serial log-transformed values. Pearson’s correlation analysis was used to determine relationship between longitudinal changes in these biomarkers.Finally, for HIV-infected patients who underwent serial CSF collections, each individual’s biomarker levels from the follow-up visits were divided by his or her baseline CSF levels to determine fold-change in IL-8, IP10, p-Tau