Results also show that the sensor has a fast response time of 920 ms.A high sensitivity and easily fabricated liquid level sensor based on the V-groove structure plastic optical fiber (POF) was described. In the design, the V-groove structure on the POF is produced by using a die-press-print method, which effectively reduces the complexity of the fabrication process and makes it easier for mass production of liquid level sensors. This greatly enhances the usefulness of the proposed sensor in cost effective liquid level sensing applications. The transmission characteristic of the POF could be changed when the V-groove structure was immerged or emerged by the rising or falling liquid. The liquid level sensing performances for the sensor probes with different structural parameters were investigated, and the sensor performances for the liquids with different refractive indices and the sensor dynamic response were also tested. Experimental results show that the sensor’s sensitivity can reach 0.0698 mm Liquid level measurements play an important role in various applications, such as fuel-level detection in tanks, liquid level monitoring for the storage and transportation of chemical products, and leakage monitoring in medical instruments and industrial process, etc. To date, many kinds of liquid level sensing techniques have been reported, including electrical and optical technologies. Compared with traditional electrical counterparts, the optical liquid level sensor is a safer option as they do not generate electric sparks at work and can resist the corrosion of chemical reagents. Thanks to the development of optical fiber technology in recent decades, optical fibers can be designed as liquid level sensors ,2,3,4. ORecent development of plastic optical fibers (POFs) makes them available for sensing fields ,10. CompIn this paper, we present a high sensitivity and simple liquid level sensor by using a POF with a V-groove structure that was fabricated on the surface of a POF by a metal mould using a simple die-press-print method. When the V-groove structure on the POF is immerged or emerged by the rising or falling liquid, the coupling characteristics of the transmitted light will be altered, which will lead to a changed output optical power. The changes of the liquid level can be measured by monitoring the POF output optical power variations. The proposed sensor is compact, has a simple structure, is easy to fabricate, and its resolution can be set easily by changing the V-groove structure parameter. These make it very attractive for liquid level sensing applications requiring compact and low cost solutions.−4 °C−1, while the cladding material is a fluorinated polymer with a thickness of 10 μm, a lower refractive index of 1.41, and thermo-optic coefficient of −3.50 × 10−4 °C−1. The POF sensor probe was fabricated by a simple mechanical die-press-print method, as shown in A commercial step-index POF  was used to fabricate the sensing probe. The core material of the POF is polymethyl methacrylate (PMMA) with a diameter of 980 μm, refractive index of 1.49, and thermo-optic coefficient of −1.15 × 10The V-groove structure on the POF will cause the higher order propagating modes to refract into the cladding and radiate out of the fiber at one side of the V-groove structure, while some of them could couple back to the POF at the other side of the structure. The power of coupled light is dependent on the medium’s refractive index and the structural parameters of the V-groove. When all the V-groove structure is initially exposed in air whose refractive index is low, the light coupling between every V-groove structure is weak, thus the output power at the POF end is low. As the liquid level increases, the V-groove structure will be filled with the liquid, and the refractive index of the medium in the V-groove region increases. As a result, the light coupling is not so weak any more, and the output light power is enhanced accordingly. This can be explained as follows.α is the incident angle of a light ray at the core-cladding interface of the POF, β is the V-groove angle, and inθ is the incident angle at the V-groove region. It is found that the incident angle at the V-groove region is decreased, which will lead to more light power to go into the cladding or radiate out of the fiber. For a light ray that transmits from the POF cladding into the V-groove region medium (the polarization is ignored), the transmission coefficient, T, can be expressed as , is the corresponding refraction angle at the cladding-medium interface in the V-groove region, and exn is the refractive index of the external medium in the V-groove region. It can be found that the transmission coefficient, T, is expressed as a function of the incident angle, inθ′, and the refractive index of the external medium, exn.For one V-groove structure (sensing area) situation, as shown in essed as :(1) T=0.T and exn ranging from 1 to 1.41 for different incident angles. It is found that the value of T increases when the incident angle decreases at the cladding-medium interface, and it is also found that as the refractive index of the external medium in the V-groove region increases, T increases. If the refractive index of the surrounding medium is low , some of the light power could still be guided in the fiber cladding, in this case, the value of T decreases. While for the surrounding medium with a higher refractive index, for example 1.41, which is the same as the refractive index of the fiber cladding, all the modes guided through the cladding are refracted outside the fiber, so T increases. The situation is the same for the light coupled from the V-groove region into the POF. The liquid in the V-groove region could decrease the reflection at the external medium-cladding interface and make more light power couple back to the POF. Thus, when the V-groove structure is immersed by the rising liquid, whose refractive index is higher than that of air, more light power could be transported from one side of the V-groove structure to the other side, which makes the output power increase accordingly. Thus, it is possible to measure liquid-level changes by employing the probe with a cascading V-groove structure. The total output power of the POF probe is the sum of all the light rays propagated in the initial POF, which refract into the fiber cladding, undergo coupling at all the V-groove regions, and finally reach the last POF output end. Of course, some of the light rays will be directly lost in the V-groove regions.C, where C is the volume concentration of the glycerin. The experiment was performed at a room temperature of 25 °C.i/P0, where Pi and P0 are the output power measuring in the glycerin solution and in air, respectively. The sensitivity is determined as the ratio of the normalized output power variations to the corresponding liquid level changes. The liquid level position before the first V-groove structure was set as the zero-level position. From the experimental results, it was found that, when the V-groove structure was immerged by the liquid, the output power increased, while as the liquid level changed at the straight POF sections, it remained stable; thus, the response curves of the sensor probes appear as a step ladder with rising liquid levels.The influences of the V-groove depth and pitch of the POF probes on the liquid level sensing performances were investigated, as shown in The sensitivity results from −1. This may be because more light power will be modulated for the probe with a V-groove angle of 90°.The influence of the V-groove angle on the liquid level sensing performances was investigated, as shown in We made the testing liquid level rise and fall repeatedly to examine the sensor dynamic performance. The V-groove pitch, depth, and angle of the sensor probe were 1 mm, 100 μm, and 60°, respectively. The refractive index of the testing liquid was 1.39. The output powers were logged continuously when the liquid level rose or fell, as shown in t/P25, where Pt and P25 are the output power measuring the temperature of t °C and 25 °C, respectively. The experiment results showed that as the temperature increases, the sensor output decreases. It is also found that the more the V-groove structure was immerged, the more obvious the temperature effect was, and when all the V-groove structure was immerged by the liquid, the sensitivity to temperature was 0.0007 °C−1, which was about two orders lower than the one to the liquid level. This is because the influence of temperature on the sensing performance can be divided into two aspects [The temperature effect of the sensor was also tested, as shown in  aspects . One is −1 for liquid with a refractive index of 1.39. The resolution of the proposed sensor was determined by the V-groove pitch. The sensor performance for liquids with different refractive indexes and the sensor dynamic response were also tested. The results demonstrated that the sensor sensitivity and response were similar for mediums with a refractive index ranging from 1.36 to 1.39, and a fast response time of 920 ms was obtained. Finally, the temperature dependence was analyzed, when all the V-groove structure was immerged by the liquid, the sensitivity to temperature was 0.0007 °C−1, which was about two orders lower than the one to the liquid level. Therefore, it is easy to distinguish the responses of temperature and liquid level changes. The sensor is a low-cost solution for detecting the liquid level, and its fabrication method can be used for other sensing applications.We have presented a high sensitivity and easily fabricated liquid level sensor based on a POF with a V-groove structure. The sensing probe was fabricated by the die-press-print method, which effectively reduces the complexity of the fabrication process and makes it easier for mass production of liquid level sensors. This process increases the liquid level sensitivity of the sensor. The influence of the structural parameters, such as the V-groove pitch, depth, and angle, on the sensing performance were investigated experimentally. Experimental results showed that the sensitivity could be improved by changing the structural parameters. When the V-groove pitch was 2.5 mm, the V-groove depth was 300 μm, and the V-groove angle was 90°, the sensitivity reached 0.0698 mm


IntroductionThe impact of donor age on liver transplantation is well known. Data on an appropriate donor age cut-off for living donor liver transplantation (LDLT) with a background of hepatitis C (HCV) is generally limited. The objective of this study was to determine whether limiting donor age to less than 35 years improved outcomes in patients with HCV-related end-stage liver disease (ESLD).MethodsThis was a retrospective review of 169 patients who underwent LDLT for HCV-related ESLD. The patients were divided into two groups based on whether they received grafts from donors ≤ 35 (Group 1) or > 35 (Group 2) years of age. Kaplan Meier curves were used to determine survival. Uni and multivariate analysis were performed to determine independent predictors of mortality.ResultsMean donor age was 25.1 ± 5.2 and 40.1 ± 3.4 years (P < 0.0001). Early allograft dysfunction (EAD) was seen in 11.7% patients in Group 1 versus 29.6% in Group 2 (P = 0.02). A significant difference in mortality was present between the two groups, i.e., 33.3% versus 15.8% (P = 0.04). The estimated four-year overall survival (OS) was 78% and 64% (P = 0.03). Upon doing univariate analysis, the donor age (P = 0.04) and EAD (P = 0.006) were found to be significant variables for mortality. On multivariate analysis, EAD was the only independent predictor of mortality .ConclusionOpting for younger donors (≤ 35 years) for HCV-related ESLD patients lowers the risk of EAD and improves overall survival. Living donor liver transplantation (LDLT) is an invaluable therapeutic modality in regions with deceased donor organ shortage . SeveralWe have been very cautious in our donor selection in terms of donor age compared to other programs . The ageThis was a retrospective review of patients who underwent LDLT. Until April 2016, 303 LDLTs were performed at the Shifa International Hospital, Islamabad. A total of 169 patients who underwent LDLT for HCV-related ESLD were included in the study. Patients with acute liver failure, age less than 18 years, dual graft, domino recipient, and those transplanted after 30th October 2015 were excluded.The details of the donor and recipient evaluations and selection have been reported elsewhere , 12. DonThe donor/recipient characteristics and operative variables were assessed. For this study, the patients were divided into two groups based on donor age; Group 1 had grafts from donors aged 35 years and below and Group 2 had donors aged 36 years and above. Donor characteristics were compared between the two groups including gender, body mass index (BMI), and graft and operative variables. A comparison was also made for recipient characteristics including age, gender, BMI, child Turcot Pugh score (CTP) score, model for end-stage liver disease (MELD) score, the presence of hepatocellular carcinoma (HCC), and operative variables. In terms of outcomes, the following variables were assessed; 1) early allograft dysfunction (EAD); 2) HCV recurrence; 3) biliary complications; 4) 90-day mortality; 5) overall mortality. Biliary complications were categorized based upon Clavien Dindo grading . EAD wasFor categorical variables, the Chi-Square test and the Fisher exact test were used, while for interval variables, independent tests were applied. For survival, Kaplan Meier curves were generated, and log rank tests were used to determine significance. Overall survival was calculated by subtracting date of death/last follow up from date of transplant. A Cox proportional hazard model was used to determine independent predictors of patient survival. Variables with a P-value < 0.1 were considered for univariate analysis and significant variables (P < 0.05) on univariate analysis were included in the multivariate analysis. All analysis was performed on Statistical Package for Social Sciences (SPSS) version 20 . The study was approved by the hospital ethics committee.Donor and recipient characteristicsTable Comparison of outcomesNo significant differences were observed between the two groups in terms of biliary complications and HCV recurrence. There was a statistically significant difference in EAD and overall mortality as shown in Table Predictors of survivalEstimated one-year overall survival (OS) for the whole group was 87%. Estimated one-year OS for Groups 1 and 2 were 88% and 71% and estimated four-years OS were 78% and 64% and was significantly different P = 0.03)  conferred survival advantage secondary to the lower rates of EAD. Variable donor age cut-offs have been used in different studies. This represents differences in the unique genetic makeup of the individual population, etiology of liver failure, and DDLT versus LDLT -18. A nuSeveral prognostic factors have been proposed to identify the risk factors for graft loss after transplantation . ImportaThe current study demonstrates superior survival with donors ≤ 35 years in a relatively younger donor cohort (maximum age 45 years). The rate of biliary complications in our cohort was similar between the two groups probably because biliary complications have been associated with relatively older and extended criteria donors . Donor aWithout much impact on other outcome variables, the younger donor age had a significant impact on the overall survival of transplanted patients. Donor gender and BMI were the only variables that were unevenly distributed between the two groups but did not impact survival on univariate analysis. In order to confirm the impact of gender, we also looked for the gender-specific rate of EAD and mortality in recipients of grafts ≤35 years of age and found no significant difference. Several mechanisms have been proposed to explain changes in liver architecture with increasing age. Perhaps the most widely understood is telomere shortening, which is most active in the 30s -27. SimiThe current study identified EAD as an independent predictor of mortality and was seen more frequently in an older donor age (>35 years). The Survival for patients who received grafts from younger donors was 88% versus 71% at one year. Clearly, one-year survival of 71% is below the international average, but an overall one-year survival of 87% for HCV-related ESLD is very much in line with international recommendations .The current study demonstrates a major reduction in the risk of death post-transplantation in the group with donors < 35 years age secondary to lower rates of EAD. The ideal donor age cut-off remains debatable due to the interplay of multiple donor, recipient, and treatment-related variables that impact graft dysfunction. It is unclear if larger studies with longer follow-up can better answer this question, but they can definitely enhance our understanding of this important variable and how it relates to patient outcomes.


RNA isolated from CSF of patients with Alzheimer´s disease (AD) and from control patients with frontotemporal lobe dementia (FTLD), major depressive episode (MDE) without clinical or neurobiological signs of AD, and healthy individuals, was reverse transcribed with primers against nine let-7 family members, and miRNAs were quantified and analyzed comparatively by quantitative PCR. let-7 miRNAs were present in CSF from patients with AD, FTLD, MDE, and healthy controls. However, the amount of individual let-7 miRNAs in the CSF varied substantially. CSF from AD patients contained higher amounts of let-7b and let-7e compared to healthy controls, while no differences were observed regarding the other let-7 miRNAs. No increase in let-7b and let-7e was detected in CSF from FTLD patients, while in CSF from MDE patients, let-7b and let-7e copy levels were elevated. In CSF from AD patients, let-7b and let-7e were associated with extracellular vesicles. let-7 family members present in the CSF mediated neurotoxicity in vitro, albeit to a variable extent. Taken together, neurotoxic let-7 miRNAs are differentially and specifically released in AD, but also in MDE patients. Thus, these miRNAs may mirror common neuropathological paths and by this serve to unscramble mechanisms of different neurodegenerative diseases.MicroRNAs (miRNAs) are non-coding RNAs originally involved in RNA silencing and post-transcriptional regulation of gene expression. We have shown in previous work that the miRNA let-7b can act as a signalling molecule for Toll-like receptor 7, thereby initiating innate immune pathways and apoptosis in the central nervous system. Here, we investigated whether different members of the miRNA family  Alzheimer´s disease (AD) is a progressive neurodegenerative disease and is considered to be the most common cause of dementia. Clinical symptoms of AD are characterized by deterioration of cognitive abilities due to hippocampal and whole brain atrophy . Initiallet-7 miRNA family comprises nine mature members with strong sequence similarity, which are highly conserved across species [Materials and Methods). Let-7 family members belong to the most abundantly expressed miRNAs in the brain [MicroRNAs (miRNA) are non-coding RNAs that are involved in posttranscriptional regulation of gene expression. They bind to the 3´ untranslated regions of their target mRNAs, leading to repression of translation or to mRNA degradation. The human  species . Variatihe brain . They arhe brain  that mayhe brain –8.Given the miRNAs’ stability in body fluids and the possibility to detect low concentrations of these molecules by quantitative PCR, miRNAs make attractive targets in the search of novel biomarkers for neurodegenerative diseases. Variations in miRNA copy numbers in the CSF of patients with various CNS diseases, such as Alzheimer’s disease (AD) , traumatlet-7 family in the CSF of AD patients. For comparison, CSF from healthy controls and from patients with other CNS diseases associated with neurodegeneration and/or cognitive impairment such as frontotemporal lobe dementia (FTLD) and major depressive episode (MDE) [In this study, we systematically quantified members of the de (MDE)  were anag, room temperature, 10 min), aliquoted (250 μl), and frozen within 30 min after lumbar puncture. The material was stored at –80°C and was at no time thawed/refrozen. Sampling and storage conditions were identical for samples from all patient groups.The study was approved by institutional review boards , as described previously [g for 10 min. The aqueous phase was mixed with 1.25 volumes of 100% ethanol. After sample application to a filter cartridge, washing was performed according to the manufacturer's instructions. RNA was eluted in 100 μl of RNAse-free water. RNA was quantified by nanophotometry (Implen).Total RNA was isolated using the eviously . Briefly30–40 ng of total RNA of CSF samples from AD, FTLD, and MDE patients as well as controls were reverse transcribed using the TaqMan MicroRNA Reverse Transcription Kit (Applied Biosystems). In case of miRNA isolation from exosomes 5 μl RNA were used. MiRNA levels expressed as CT-values were determined by real time PCR (qPCR) using TaqMan MicroRNA assay for hsa-let-7a, hsa-let-7b, hsa-let-7c, hsa-let-7d, hsa-let-7e, hsa-let-7f, hsa-let-7g, hsa-let-7i, hsa-miR-98, hsa-miR-16, hsa-miR-24, and hsa-miR-124 according to the manufacturer's instructions on a 7500 Fast Real Time PCR system (Applied Biosystems). Expression levels of miRNAs in the CSF from one individual patient were determined in parallel on one single PCR plate. Quantitative miRNA levels expressed as absolute copy numbers were determined using TaqMan MicroRNA assay on a StepOnePlus Real Time PCR system (Applied Biosystems). Copy numbers were calculated implementing a standard curve using synthetic oligoribonucleotides and plotting CT-values versus copy number, a CT-value of 40 corresponding to a copy number of 0, as described previously . qPCR wag for 30 min at 4°C. One volume of exosome isolation reagent was added to the supernatant and mixed by vortexing before incubating for 1 h at 4°C. Samples were centrifuged at 10,000 g for 1 h at 4°C. Supernatant was aspirated. The complete volume of supernatant was used for RNA isolation. The pellet containing extracellular vesicles was re-suspended in either 200 μl PBS  or 20 μl ice-cold Exosome Resuspension Buffer (for western blotting).Isolation of extracellular vesicles from CSF was performed with the Total Exosome Isolation Kit according to the manufacturer’s instruction (Life Technologies). Briefly, 200 μl (for RNA isolation) or 400 μl (for western blotting) of CSF were thawed on ice and centrifuged at 10,000 Western blotting was performed, as previously described . Anti-Fl5′-UGAGGUAGUAGGUUGUAUAGUU-3′; let-7b, 5′-UGAGGUAGUAGGUUGUGUGGUU-3′; let-7c, 5′ UGAGGUAGUAGGUUGUAUGGUU-3′; let-7d, 5′-AGAGGUAGUAGGUUGCAUAGUU-3′; let-7e, 5′-UGAGGUAGGA GGUUGUAUAGUU-3′; let-7f, 5′-UGAGGUAGUAGAUUGUAUAGUU-3′; let-7g, 5′-UGAGGUAGUAGUUUGUACAGUU-3′; let-7i, 5′-UGAGGUAGUAGUUUGUGCUGUU-3′; mutated oligoribonucleotide (mut. oligo), 5′-UGAGGUAGAAGGAUAUAAGGAU-3′.The following native oligoribonucleotides were synthesized by Purimex DNA/RNA-Oligonucleotide, Grebenstein, Germany: let-7a, Cortical neurons were generated from C57BL/6 mice and cultured, as described previously . SH-SY5YVarious doses of oligoribonucleotides and other reagents were added to cell cultures for various durations, as indicated. Imiquimod and DAPI were obtained from InvivoGen and Roche, respectively. Terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) staining of cells was conducted using the In Situ Cell Death Detection kit, TMR red (Roche). For each condition, experiments were performed in duplicates.Immunostaining was performed, as described previously . NeuN, nNeuN-positive cells were quantified by analyzing six high-power fields per coverslip. Numbers of NeuN-positive cells observed for each condition were compared with control conditions. Results were expressed as relative neuronal viability. TUNEL-positive cells were quantified in six high-power fields per coverslip and were expressed relatively to NeuN-positive cells in the same high power field as fold change to negative control.TM Kit (BD Biosciences), and stained with anti-TLR7 Alexa488 antibody (Imgenex). An Alexa 488-conjugated isotype served as negative control. Data were collected on a BD FACSCanto II and analyzed by FlowJo Version 9.5.2.SH-SY5Y cells were fixed, permeabilized using the BD Cytofix/Cytopermt test. Statistical differences over multiple groups of individuals or patients were determined using the one-way ANOVA test followed by Sidak’s multiple comparison post hoc test of patient group vs. control group. Adjusted p-values for patient and control groups were determined by ANCOVA with gender and age as covariates. Statistical differences over all tested conditions in vitro were determined using the Kruskal-Wallis test followed by Dunn´s Multiple comparison post hoc test of total vs. fraction or negative control vs. treatment. Differences were considered statistically significant when p < 0.05.Data are expressed as indicated in the figures. Statistics as indicated in the figure legends were calculated using GraphPad Prism version 5.0 for Mac OS X . Otherwise, the statistical software SPSS 24 was used. In detail, data were described as mean or median and standard deviation. Statistical differences between two groups of individuals or patients were determined by the Student’s let-7-related miRNA miR-98, miR-124, which is abundantly and specifically expressed in the brain [let-7 miRNAs were present at different copy levels in different individuals, expression patterns were similar in AD patients and healthy controls. In particular, let-7a, let-7b, let-7c, and let-7e were found at high copy levels, while let-7d, let-7f, let-7g, let-7i, and miR-124 were present at lower copy levels in the majority of samples or were only marginally detected  in both experimental groups , while it was detected in only 3 out of 8 CSF samples from FTLD patients. For comparison, let-7d was present in 6 out of 12 CSF samples from AD patients and in 3 out of 10 from healthy controls. CT-values for miR-124 expression were elevated in CSF from FTLD patients compared to all other tested groups  cannot reliably distinguish between different specific extracellular vesicle species such as exosomes or between extracellular vesicles and protein aggregates, such as protein-miRNA complexes [let-7 miRNAs bind to TLR7 only when associated with extracellular vesicles that are released during neuronal injury. Thereby, these oligonucleotides may engage TLR7 in the endolysosomal compartment of neighboring neurons and mediate injury, whereas cytosolic miRNAs do not mediate injury via TLR7 in intact neurons.We showed in previous work that intrathecal let-7b, preferentially expressed in neurons and released from injured cells, induces further neurodegeneration via endolysosomal TLR7, a GU-rich sequence motif within the oligoribonucleotide being essential for receptor activation . The difnal loss . Based otivation , and somomplexes . Neverthlet-7 miRNAs were detected in the CSF from all patient groups. In particular, let-7b and let-7e were consistently present at high expression levels compared to the other miRNAs. Even if statistical analysis was adjusted for gender and age, let-7e was significantly elevated in AD and MDE CSF. In terms of let-7b, adjusted statistical testing pointed to a potential interaction between gender and AD, provided that additional male individuals are included in the study. In contrast to let-7b that induced profound neurodegeneration in vitro, let-7e mediated only mild neuronal injury. Likewise, let-7a and let-7c were detected at high copy levels in CSF from patients and controls, but exhibited only minor neurotoxic effects. Conversely, let-7f and let-7g mediated distinct neurodegeneration in vitro but copy numbers were low in all CSF samples. Thus, the capacity of inducing neurotoxicity in vitro did correlate with high copy numbers in the CSF in the case of let-7b, but this relation was not observed for the other let-7 miRNAs. These findings may reflect distinct biological functions and potential of each let-7 miRNA in its extracellular form.In this study expression levels of miRNAs in human CSF were determined for one individual patient on one PCR plate to compare copy levels of different miRNAs within one AD, FTLD, or MDE patient or within one control subject. Similar expression patterns of different While RNA quantities isolated from CSF did not relevantly differ between AD patients and controls, it did vary between controls and FTLD as well as MDE patients. Therefore, equal RNA amounts were applied for reverse transcription to standardize the experimental procedure. We determined copy numbers of the selected miRNAs by utilizing dosage curves of the respective synthetic miRNA and compared miRNA expression levels in the CSF with concentrations of the respective synthetic oligoribonucleotide. We report here on an increase of let-7b copies in the CSF from AD patients, thereby confirming our previous studies in which different patient cohorts were used . In addiA previous study reported on increased let-7f amounts in AD CSF . In the let-7 miRNAs were detectable in sufficient amounts in the AD CSF may be due to specific packaging of miRNAs into extracellular vesicles such as exosomes and release into the CSF. miRNAs, which may be protected by their exosomal carriers, feature a major stability in CSF and are therefore considered as potential biomarkers for CNS diseases [let-7 miRNAs in AD CSF, which had been stored for extended periods . qPCR analysis revealed slightly reduced, but readily detectable miRNA copy levels  or patients with AD  were assayed by qPCR using primers specific for miR-98, miR-24, or miR-16. Data are presented as box-and-whisker-plots of mean CT-values performed in triplicates with median for each miRNA.CSF samples from healthy controls ((TIF)Click here for additional data file.S2 Fign = 10–11, AD-1 –AD-6; AD-8 –AD-12) and healthy controls  were assayed by qPCR using primers specific for let-7a, let-7b, let-7c, let-7e, or let-7g and were normalized to the standard of the respective synthetic miRNA. Statistical analysis was performed using unpaired t-test, and p-values were adjusted for age and gender by ANCOVA.CSF from patients with AD ((DOCX)Click here for additional data file.S3 Fign = 10, Co-1 –Co-10) and from patients with FTLD  or MDE  were assayed by qPCR using primers specific for let-7b or let-7e and were normalized to the standard of the respective synthetic miRNA. Statistical analysis was performed using one-way ANOVA followed by Sidak’s multiple comparison post hoc test of the respective patient group vs. control group. p-values were adjusted for age and gender by ANCOVA.CSF from healthy controls ((DOCX)Click here for additional data file.S4 Fign = 10, Co-1 –Co-10) were assayed by qPCR using primers specific for let-7b or let-7e and were normalized to the standard of the respective synthetic miRNA. Data are presented as mean ± SD. Each patient resembles one dot. Statistical analysis was performed using paired Student’s t-test.CSF from healthy controls depicted in (TIF)Click here for additional data file.S5 Fign = 4), and both pellet of EV and supernatant (S/N) obtained during the isolation procedure were subjected to western blot using flotillin-1 (A) or CD63 (B) Abs. Neurons: Cortical neurons isolated from C57BL/6J mice.Extracellular vesicles (EV) were isolated from CSF of AD patients ((TIF)Click here for additional data file.S6 Figvs. treatment. P values in (B) depicting the dose response: let-7f **p = 0.0033, let-7g **p = 0.0018; time response: let-7f **p = 0.0011, let-7g ***p = 0.0005. P values in (C) depicting the dose response: let-7f *p = 0.0223, let-7g **p = 0.0042; time response: let-7a ***p = 0.0008, let-7f ***p = 0.0009, let-7g ***p = 0.0005. #p<0.05, ##p<0.01, ###p<0.005. mut. oligo, mutated oligoribonucleotide. (D) Neurons were incubated with 10 μg/ml of the respective miRNA for 4 days. Subsequently, they were immunostained with NeuN Ab (green) and stained by TUNEL assay (red) and with DAPI (blue). Scale bar 50 μm.(A) Neurons from C57BL/6 mice were incubated with 5 μg/ml of the respective oligoribonucleotide or with 5 μg/ml imiquimod, which served as a positive control for TLR7-mediated neuronal cell death, for 5 days. Subsequently, cells were fixed and immunostained with neurofilament Ab (red), MAP-2 Ab (green), and stained with DAPI (blue). Scale bar 100 μm.  Neurons from C57BL/6 mice were incubated with indicated concentrations of let-7a, let-7c, let-7d, let-7f, let-7g, let-7i, or a mutated control oligoribonucleotide for 4 days, or with 10 μg/ml of the respective miRNA for indicated time points. 10 μg/ml imiquimod served as a positive control. Cells were immunostained with NeuN Ab, stained by TUNEL assay, or with DAPI. Each condition was performed in duplicate and averaged. NeuN-positive cells were quantified and were expressed as relative neuronal viability. TUNEL-positive cells were quantified, set in relation to NeuN-positive cells, and were expressed as fold-change. Mean ± SD from 3–4 individual experiments, Kruskal-Wallis test followed by Dunn´s Multiple comparison post hoc test of negative control (TIF)Click here for additional data file.S7 FigSH-SY5Y cells were fixed, permeabilized, stained with anti-TLR7 Alexa 488 Ab, and analyzed by flow cytometry. An Alexa488-conjugated isotype was used as a negative control.(TIF)Click here for additional data file.S8 Fign = 10) that had been stored for 85,1+/-29,4 (mean ± SD) months after lumbar puncture at -80°C, were assayed by qPCR using primers specific for let-7b, let-7e, or miR-124. Data are presented as box-and-whisker-plots of mean CT-values performed in triplicates with median for each miRNA.CSF samples from patients with AD ((TIF)Click here for additional data file.


We compared in vitro microPET assessed 18F-NaF uptake between culprit and non-culprit human carotid plaques. Furthermore, we compared 18F-NaF uptake with calcification visualized on microcomputed tomography (microCT).18F-NaF and scanned using a microPET and a microCT scan. The average PET assessed 18F-NaF uptake was expressed as percentage of the incubation dose per gram (%Inc/g). 18F-NaF PET volume of interest (VOI) was compared with CT calcification VOI.Carotid plaques from stroke patients undergoing surgery were incubated in 18F-NaF uptake in culprit carotid plaques was comparable with the uptake in non-culprit carotid plaques . Only a median of 10% (IQR 4 to 25) of CT calcification VOI showed increased 18F-NaF uptake, while merely a median of 35% (IQR 6 to 42) of 18F-NaF PET VOI showed calcification on CT.23 carotid plaques  were included. The average 18F-NaF PET represents a different stage in the calcification process than CT. We observed a similar PET assessed 18F-NaF uptake and pattern in culprit and non-culprit plaques of high-risk patients, indicating that this method may be of more value in early atherosclerotic stenosis development.The online version of this article (10.1007/s12350-018-1325-5) contains supplementary material, which is available to authorized users. Surgical removal of atherosclerotic plaques from the carotid artery highly reduces the risk of future stroke in symptomatic patients with ≥ 70% stenosis.In order to improve risk stratification, research has been focused on the identification of plaques at risk for rupture, so-called vulnerable plaques.1018F-sodium fluoride (18F-NaF) positron emission tomography (PET) for imaging of atherosclerotic plaques.1318F-NaF predominantly binds to areas of microcalcification within the plaque.20Recently, several studies have shown the feasibility of 18F-NaF may additionally be a useful marker for plaque vulnerability.18F-NaF uptake than non-culprit and low-risk coronary plaques.18F-NaF uptake in carotid plaques are limited and their usefulness for the prediction of future stroke is unclear.27It has been suggested that 18F-NaF uptake between culprit and non-culprit carotid plaques from stroke patients, using non-macrocalcified renal arteries from healthy kidney donors as negative controls. The secondary objective is to compare the distribution of 18F-NaF uptake on microPET with calcification visualized on a high-resolution microcomputed tomography (microCT) in carotid plaques.The primary objective of this study is to compare in vitro microPET assessed Carotid plaques were collected from stroke patients who underwent carotid endarterectomy (CEA) at the Department of Surgery (Division of Vascular Surgery) of the University Medical Center Groningen (UMCG), between July 2015 and March 2016. Indication for CEA was decided by a surgeon expert panel and was based on the presence of symptomatic (culprit) stenosis (≥ 50%) or asymptomatic (non-culprit) stenosis (≥ 70%) of the internal carotid artery, according to internal guidelines.In order to increase the reliability of our measurements, we used renal artery specimens from healthy kidney donors as negative controls. The specimens were obtained during living donor nephrectomy.Clinical and demographic data from the included patients were collected from medical records. In the group with culprit plaques, medication use and history of cardiovascular diseases prior to the recent event were registered. The study was reviewed by the ethics committee of the UMCG (METc 2015/258). All patients gave written informed consent.18F-NaF in 20 mL. After incubation, the plaques and renal arteries were carefully rinsed 5 times with 10 mL PBS. Then, tissue samples were weighed and microPET and microCT scans were performed. After the imaging procedure, the carotid plaques were cut transversely into segments of 3 to 4 mL. The renal arteries had a maximum thickness of 5 mL and therefore no cross-sections were made. The segments were embedded in paraffin for histological analysis.Immediately after excision, carotid plaques and renal artery specimens were placed into phosphate buffered saline (PBS) and kept on ice. Both were incubated for one hour in 49.4 ± 7.2 MBq 18F-NaF was produced by passing a solution of 18F-fluoride in water over a quaternary methyl ammonium (QMA) light anion exchange cartridge . After washing the QMA with water, [18F]-fluoride was eluted with saline and passed over a sterile Millex GS 0.22 µm filter . The radiochemical purity for all runs was > 95%.Carotid plaques and renal arteries were positioned into a microPET scanner , and an emission scan of 30 minutes was performed. After the PET scan was finished, the bed of the PET scanner was transferred to a microCT scanner  without moving or touching the tissue samples. The CT exposure settings were 50 keV and 500 µAs, and a 100-ms exposure time for 360 projections during one 360° rotation.The PET scans were reconstructed into a single frame of 30 minutes, using OSEM2D (4 iterations and 16 subsets), after being normalized and corrected for attenuation and decay of radioactivity. The CT images were reconstructed with the Feldkamp algorithm.3018F-NaF uptake, three-dimensional volumes of interest (VOIs) were drawn around the whole tissue samples. The uptake (in kBq/cc) was corrected for weight of the specimen and the incubation dose, and expressed as percentage uptake of total incubation dose per gram of tissue (%Inc/g). It was assumed that 1 cubic centimeter equals 1 gram of tissue.The PET and CT images were automatically registered using PMOD 3.7 . The registration was visually inspected and manually corrected when necessary. For quantification of the average 18F-NaF PET areas with a threshold of ≥ 50% of the maximum 18F-NaF uptake and assigned as 18F-NaF PET VOI. VOIs were automatically drawn around CT areas with a Hounsfield Unit (HU) ≥ 1000 and assigned as CT calcification VOI. The threshold of 50% of the maximum uptake value was chosen in order to select the volume with the highest 18F-NaF uptake, and thereby minimize the bias of a partial volume effect.18F-NaF PET VOIs and CT calcification VOIs, an intersection VOI was automatically drawn. Then, the CT calcification area (HU ≥ 1000) within the 18F-NaF PET VOI was measured and expressed as a percentage of the 18F-NaF PET VOI; and the other way around; 18F-NaF PET uptake area (≥ 50% of maximum 18F-NaF uptake) within the CT calcification VOI was measured and expressed as a percentage of the CT calcification VOI.VOIs were also automatically drawn around 18F-NaF uptake. The pattern of 18F-NaF uptake on PET images was compared with the results of histology. Furthermore, the two renal arteries with the highest 18F-NaF uptake were selected for staining (negative controls). Only negligible 18F-NaF uptake was expected and no calcification in the renal arteries, because only healthy kidney donors with a renal vasculature without any signs of atherosclerosis are eligible for transplantation.To validate our data, von Kossa and alizarin red stainings for calcification were performed on two plaque segments without any CT calcification, but with clear To obtain further information on the vulnerability of included carotid plaques, segments of five culprit and three non-culprit plaques were assessed for histological features of vulnerability. The segments were stained with Martius Scarlet Blue (MSB) using histochemistry and for CD68- and CD34-expressing cells using immunohistochemistry.U test (two groups) or the Kruskal–Wallis test (≥ two groups) was used. Categorical data were analyzed with the Chi Square test. A Spearman Correlation was used to test the association between continuous data. A two-sided P-value ˂ 0.05 was considered statistically significant. Statistical analyses were performed using SPSS for Windows (version 23.0).Descriptive data are presented as frequencies (percentage), median (interquartile range), or mean ± SD. Based on the distribution of data , differences between data were analyzed with non-parametric tests. For continuous data the Mann–Whitney P = 0.020). The mean time between the cerebrovascular event  and CEA in the group with culprit plaques was 21 ± 14 days. All patients in the non-culprit group had a history of stroke related to the contralateral carotid artery. The time range between that stroke and the recent non-culprit CEA was 2 to 23 months. The healthy kidney donors  were younger than CEA patients (P = 0.001) and had no history of cardiovascular disease.We included 23 carotid plaques (17 culprit and 6 non-culprit) from 23 patients  who had undergone CEA, and 15 renal artery specimen from healthy kidney donors .PET images of all 23 plaques, showed a heterogeneous 18F-NaF uptake was similar in culprit and non-culprit carotid plaques , while the uptake in carotid plaques was significantly higher than in renal arteries  , consisting of median 6% (IQR 3 to 10) of the total plaque volume (Table 18F-NaF in the 18F-NaF PET VOIs was median 4.7 (IQR 3.9 to 6.2) %Inc/g , consisting of median 16% (IQR 3 to 27) of the total plaque volume   was lower than that of the CT calcification VOIs (median 3258 HU [IQR 2465 to 3616]), as these 18F-NaF PET VOIs consist only partially of CT assessed calcification (Table The averaged HU of the  HU IQR 9 to 1349)18F-NaF uptake of the plaque (%Inc/g) and the CT calcification VOI , although 18F-NaF uptake seems to increase when CT calcification VOI increases in most plaques . Areas with negligible 18F-NaF uptake did not show any histological evidence for calcification. As expected, no calcifications were identified in the segments of the renal arteries .The von Kossa and alizarin red stainings showed calcification deposits in the two selected The five culprit and three non-culprit plaques segments showed all at least one characteristic of vulnerability. Macrophage infiltration was clearly visible in all segments, as was the presence of intraplaque microvessels, without a visual difference in culprit or non-culprit segments. Two culprit plaques and one non-culprit plaque showed a clear thrombus indicative of intraplaque hemorrhage. One non-culprit plaque showed a very thin fibrous cap .18F-NaF uptake, in culprit and non-culprit human carotid plaques. We hypothesized that 18F-NaF uptake in culprit plaques was higher than in non-culprit carotid plaques, based on previous results in coronary plaques, and the concept of microcalcification and plaque rupture.18F-NaF uptake in culprit and non-culprit carotid plaques. Interestingly, we found that 18F-NaF uptake was present in regions without evidence of calcification on CT scan. Furthermore, most of the CT calcification VOIs had low 18F-NaF uptake, confirming that both techniques represent a different stage of calcification.32The present study investigated in vitro microPET assessed 18F-NaF uptake was higher in culprit carotid artery stenosis than in the contralateral non-culprit stenosis in 18 patients with recent CVA .18F-NaF uptake reported as mean target-to-background ratio was higher in culprit plaques (2.12 ± 0.44) than in contralateral non-culprit plaques .Recently, Vesey et al. showed that in vivo 18F-NaF uptake in culprit and contralateral non-culprit plaques, the differences were small and a substantial overlap between the uptake values in both groups was present, as was in the study of Quirce et al. Furthermore, the 18F-NaF uptake between patients scheduled for CEA and control patients differed to a larger extent (delta 0.17 SUVmean) than the difference between culprit and non-culprit uptake (delta 0.07 SUVmean).18F-NaF uptake can be determined, only when compared with control patients there is a relevant difference. This is in line with the results of our study. Although the sample size is small, our data show that 18F-NaF uptake between culprit and non-culprit carotid plaques was comparable while the uptake in plaques was significantly higher than in control renal arteries.First, although Vesey et al. did find a significant difference between 18F-NaF uptake was related to the degree of stenosis on CT, but they did not report the stenosis degree in the separate groups, neither did Quirce et al. In our study, the stenosis degree in both, non-culprit and the culprit plaques, was high and all plaques showed high-risk features based on histology. This could explain the similar 18F-NaF uptake.Second, there may be differences in the degree of stenosis of the carotid arteries between patients in the aforementioned studies and our patients. Vesey et al. found that coronary plaques of patients with myocardial infarction.In contrast, Joshi et al. did find a difference between culprit and non-culprit 18F-NaF activity was increased in areas without calcification on CT and most of the CT calcification VOI showed minimal 18F-NaF uptake. This supports the idea that microcalcification, as visualized with 18F-NaF PET, and established calcification visualized on CT may reflect different stages of the calcification processes in atherosclerotic plaques.34Established calcification is a well-known marker of total plaque burden and is strongly associated with the risk for cardiovascular events.Another important example can be derived from the work of Puri et al., who performed a post-hoc patient-level analysis of 8 prospective trials in which coronary atheroma was measured with intravascular ultrasound. They showed that although statins had a clear plaque-regressive effect, they also promoted coronary atheroma calcification, indicating stabilization.3918F-NaF uptake was related to the calcium volume on CT in the majority of the carotid plaques. This is probably caused by binding of 18F-NaF at only the surface of the calcifications.18F-NaF uptake can still be found in vitro.In this study increased 18F-NaF uptake and vice versa. This suggests 18F-NaF accumulation in areas without any evidence of calcification, or at least no calcification with a size above the detection limit of the microCT scan.18F-NaF positive and CT negative segements. 18F-NaF probably binds to the relatively large surface of microcalcifications , causing an intense signal on PET images.14However, in our study a few plaques with low calcium volume had a high 18F-NaF imaging can detect biologically active plaques, before they can be visualized on CT. This implies that 18F-NaF imaging may be useful in evaluating disease progression, as was further shown in patients with aortic stenosis, where baseline 18F-NaF uptake correlated well with the calcium progression after 1 year.18F-NaF uptake in areas without established calcification on CT was the best predictor of calcium progression.These observations indicate that 18F-NaF uptake in the carotid arteries and, age and various cardiovascular risk factors in 269 patients with no history of stroke.18F-NaF imaging to identify patients at high-risk for cardiovascular disease in a low-risk population. In addition, the finding that 18F-NaF uptake in carotid plaques exceeded that of controls  and no calcification was visible in renal arteries on microCT or with histological staining adds evidence to the hypothesis that the presence of microcalcification identified by 18F-NaF is a feature of atherosclerosis. The relation between the extent of 18F-NaF uptake in bilateral carotid plaques and features of vulnerability needs to be further investigated.Furthermore, Derlin et al. found a positive correlation between 18F-NaF PET imaging with calcification visualized on microCT, this study adds knowledge to the relatively new field of 18F-NaF imaging in atherosclerosis. Our study has some limitations. First, it should be considered that the number of plaques, especially non-culprit plaques, is small and a type II statistical error might be introduced. However, given the similar distribution of 18F-NaF uptake in both groups, a high number of plaques would need to be recruited to find a statistical significant difference if present. We believe that small differences in uptake will not have any clinical implication.Strengths of our study are the inclusion of a control group, and the scanning of calcium phantoms in order to accurately determine the calcium threshold. Furthermore, by comparing calcification identified by Second, the tracer uptake could be underestimated due to partial volume effects, as with every imaging study. Third, CT images of 16 plaques (one non-culprit) out of 23 were available for analysis due to practical and technical issues. Fourth, we did not assess the plaque morphology of the right and left side in the same patient. Culprit and non-culprit plaques were derived from different patients.18F-NaF PET images and CT images are different. Clearly, 18F-NaF PET visualizes a different stage of the calcification process than CT. 18F-NaF uptake in carotid plaques exceeded the uptake in non-calcified renal arteries, but was comparable between culprit and non-culprit carotid plaques, probably due to the advanced nature of atherosclerotic disease in our patients.We have demonstrated that the calcification patterns on 18F-NaF has the potential to identify carotid plaques with active calcification. Further prospective studies on 18F-NaF uptake and symptomatology are required to assess the predictive and diagnostic value of 18F-NaF imaging in patients with early stage atherosclerosis.We conclude that Online Resource 1 (DOCX 20 kb)Online Resource 2 (JPEG 660 kb)Online Resource 3 (JPEG 109 kb)Online Resource 4 (JPEG 3228 kb)Online Resource 5 (PPTX 463 kb)Below is the link to the electronic supplementary material.


We report a human case of viral encephalitis caused by pseudorabies virus (PRV) in China. A 43-year-old man with no previous medical history presented with high-grade fever, headache and tonic-clonic seizures as well as coma. Plain computer tomography (CT) brain imaging showed hypo-density in the bilateral basal ganglia, bilateral occipital lobe, bilateral limbic lobe, and left thalamic. Next-generation sequencing (NGS) confirmed the presence of PRV in cerebral spinal fluid (CSF). Regular polymerase chain reaction (PCR) was applied to confirm the presence of PRV in the CSF and blood. In addition, serological  tests were used to further validate the presence of PRV in the peripheral blood. This case suggested that it was possible for PRV to result in human central nervous system (CNS) infection, and it is necessary for people to increase awareness of self-protection when contacting animals. Pseudorabies virus , which belongs to the Alphaherpesvirinae subfamily and genus Varicellovirus in the family Herpesviridae, causes fatal encephalitis in newborn piglets, respiratory disorders in growing-fattening pigs, and reproductive failure in sows. In the past, PRV was thought to not infect humans and just infect mammals including pigs, cattle, sheep, goats, dogs, cats, foxes, rats, and wild mice , which s2O (80–180 mmH2O). However, the patient could not provide other detailed results from CSF  and serum. On the 4th day after initial symptoms, plain CT (computer tomography) brain imaging was normal ] was preserved, for a total of 2 weeks. After 1 month of treatment, he was still dependent on tracheostomy and gastrostomy tubes, but has been weaned off mechanical ventilation. The patient was voluntarily discharged to another hospital for further rehabilitation of neurological function.The patient, a 43-year-old man with no prior medical history, who worked as a veterinarian, was admitted to the critical care department of Qilu hospital of Shandong University  due to high-grade fever of 11 days' duration, headache of 9 days' duration and tonic-clonic seizures as well as coma of 8 days' duration. His hands were punctured by a knife used during the autopsy process of dead swine 4 days before his initial symptom (fever) occurred. At the 3rd day after the initial symptoms, he developed status epilepticus and coma, requiring endotracheal intubation, treatment with intravenous midazolam as well as valproate, and he was treated in the intensive care unit (ICU) of the local hospital for 8 days. Lumbar puncture indicated an opening pressure of 230 mmHs normal . On 8th s normal . With th*109/L (3.5–9.5*109/L), platelet count 120*109/L (125–350*109/L), sodium 140 mmol/L (135–145mmol/l), potassium 3.6 mmol/L (3.5–5.5mmol/l), creatinine 89 μmol/L (62–115 μmol/l), albumin 26.3 g/L (40–55g/l), alanine transaminase (ALT) 155 U/L (9–50 U/l), and aspartate transaminase (AST) 182 U/L (15–40 U/l). Japanese encephalitis virus (JEV)-specific IgM antibody in the plasma was negative. Plain CT brain imaging showed hypo-density in the bilateral basal ganglia, bilateral occipital lobe, bilateral limbic lobe, and left thalamic  9.3 g/dL (115–150 g/dl), white cell count (WBC) 6.39thalamic . After ithalamic  was not thalamic  and 11ththalamic  after in2O (80–180 mmH2O), glucose of 1.86 mmol/L (2.8–4.5 mmol/l), protein content of 1.45 g/L (0.15–0.45 g/l), RBC (red blood cell) of 14/ml (0/ml) and WBC (white blood cell) of 189/ml (0–5/ml) (98% monocytes) on the 1st day at our hospital. An infectious disease screen of the central nervous system (CNS) using CSF included PCR against viruses , Gram and acid-fast stains, cultures and next-generation sequencing (NGS). The CSF screen was negative for current virus and bacterial infection. NGS results showed 6,198 unique sequence reads of PRV in CSF , covering 80.58% of the nucleotide sequences  of 2/ml and WBC (white blood cell) of 174/ml (70% monocytes). NGS was again performed to detect the nucleotide of PRV in CSF. However, no unique sequence reads of PRV were detected in CSF (data not shown).Lumbar puncture revealed transparent fluid with an opening pressure of 90 mmHequences . Sanger equences . Based oTo further validate our results, we sent the patient plasma and CSF from the 14th day after initial symptom to Dr. Huanchun Chen's lab  for PRV antibody testing. If the antibody titer is not higher than 0.6, the result is considered positive. PRV gB antibody (antibody titer: 0.248) in the serum was positive while PRV gE antibody (antibody titer: 1.001) in the serum was negative. However, Neither PRV gB antibody or PRV gE antibody was detected in the CSF. On the 21st and 28th days after initial symptoms, the patient's plasma was sent again to Dr. Chen's lab for PRV antibody testing. Both PRV gB antibody (separate antibody titers: 0.132 and 0.130) and PRV gE antibody (separate antibody titers: 0.557 and 0.337) in the serum were still positive.To date, there are no confirmed CNS cases of human PRV infection. There were three suspected CNS cases of human PRV infection after close contact with cats or other domestic animals . However3), typically mononuclear white and red blood cells, reflecting the hemorrhagic nature of the infectious process within brain parenchyma  count of 14, and WBC (white blood cell) count of 189/ml (98% monocytes). The clinical presentation of viral encephalitis is non-specific, including fever, varying degrees of alteration in sensorium with or without focal neurological deficits, and/or seizures. The characteristics of CSF in Herpes simplex encephalitis (HSE) include moderate pleocytosis (usually 10–200 cells/mmrenchyma , 6. The renchyma  showed tSpecific viral diagnosis could be achieved by demonstration of viral nucleic acid or antibody in CSF or isolation of the virus from CSF or brain tissue . The senVaccination is thought to be the most important method to control PRV. However, PRV vaccine is still provided for swineherds on a voluntary basis rather than as a requirement in China . PRV gE In conclusion, this case of PRV-caused encephalitis indicated that PRV could invade the CNS and infect humans. Clinicians should be especially aware that PRV-caused human encephalitis should be considered in patients with encephalitis of unknown etiology, especially those who have had a history of exposure to swine or other domestic animals. In addition, the broken skin or mucous membrane might make it easy for PRV to transmit from infected animals to human. Not only vaccinated swine but also non-vaccinated swine have the ability of transmitting PRV from pigs to human. Thus, people who worked on animal husbandry and veterinary specialty should increase awareness of self-protection when contacting diseased animals. More importantly, the outcome of PRV-caused encephalitis is poor. And there is no effective drugs to prevent the progress of the disease.We received written informed consent from the patient's wife.HY wrote the manuscript. HH, HW, and YC helped to collect the clinical data and neuroimages. HL and SD helped to send CSF and plama to Dr. Huanchun Chen's lab.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


ED) in men aged >55 years with type 2 diabetes. This prospective study included data from men aged >55 years with type 2 diabetes. ED was assessed by the International Index of Erectile Function 15‐item survey. Total testosterone (TT) levels and bioavailable testosterone were measured; the free testosterone index was calculated. Data from 155 men (aged 64 ± 7 years) were explored. The prevalence of ED and testosterone deficiency was 78.7% and 34.8%, respectively. After univariate analysis, TT and bioavailable testosterone were associated with ED (P = 0.01). After multivariate analysis, and adjustment for age, body mass index, tobacco, alcohol, duration of diabetes, TT, bioavailable testosterone, vitamin D and high‐sensitivity C‐reactive protein, we found that only high‐sensitivity C‐reactive protein was significantly predictive of ED. TT could predict ED, but it lacks specificity. We found a potential role of high‐sensitivity C‐reactive protein as a predictive marker of ED in this targeted population.To date, there is no evidence regarding the best biological marker to predict erectile dysfunction ( Men with ED had lower levels of total testosterone and bioavailable testosterone compared to men without ED but the free testosterone index was not different. Surprisingly, the hsCRP was higher in men with ED compared to the others. The multivariate analysis found a new potential role of hsCRP as a predictive marker of ED in men with diabetes, aged >55 years. Erectile dysfunction (ED) is defined as an inability to have a sufficient erection for satisfactory sexual intercourseWe carried out a cross‐sectional study including all men aged >55 years with type 2 diabetes, after oral agreement, in the Department of Diabetology of the University Hospital of Guadeloupe. Patients with renal or prostate disease and cognitive disorders were excluded.ED was evaluated with the International Index of Erectile Function 15‐item (IIEF‐15) survey. The diagnosis of ED was confirmed when the IIEF‐15 score was <26Serum TT levels were measured using an electrochemiluminescence method. HG was defined as serum TT <10.4 nmol/L. According to the Vermeulen formula, BioT was obtained from a calculation using sex hormone‐binding globulin, albumin and TT2‐test and t‐test, Pearson's and Spearman's correlation, and the odds ratio with a 95% confidence interval. Results were considered significant when P < 0.05 (two‐sided).Data are presented as the mean ± standard deviation for continuous variables, and percentages (numbers) for categorical variables. Data were analyzed using the χAfter approval by the local ethics committee, all men received a detailed note before they gave their oral informed consent. No men objected to the study.In respect to the national legislation about data protection, a declaration of conformity has been made to the National Data Information and Freedom Commission.We analyzed the data collected from 155 men aged >55 years with type 2 diabetes, included during 8 months in 2017. The characteristics of the population are summarized in Table Comparison of clinical and biological parameters between men with or without ED is reported in Table P = 0.001), and an inverse correlation between TT levels and ED (P = 0.011). This inverse correlation was not observed for BioT and FTI.BioT levels were low in 25.9% of patients. We observed a positive correlation between TT and BioT . No association was found between FTI and ED (P = 0.75). We carried out a multivariate analysis with adjustment for age, body mass index, tobacco, alcohol and duration of type 2 diabetes; we found that only TT was associated with ED . However, after additional adjustment for BioT, vitamin D and hs‐CRP, only hs‐CRP levels were associated with ED .After univariate analysis, TT and BioT were significantly associated with ED score (odds ratio [OR] 0.91, 95% confidence interval [CI] 0.84–0.98, According to our knowledge, this is the first time that the three forms of testosterone evaluation are analyzed together as predictive factors of ED.The subject of sexuality remains difficult to address, even in the field of health, which can explain why ED is not usually evaluated in men with type 2 diabetes, despite clear recommendationsAlthough the IIEF‐15 is a simple and reliable tool for ED diagnosis, it remains difficult to determine the pathophysiological origin of ED. ED observed in type 2 diabetes can result from both neuropathy and distal arteriopathyIn the present cohort, we observed 34.8% of HG, defined as a serum level of TT under 10.4 nmol/L. Our levels of TT were closer to those described in previous studiesThe use of other markers of HG, such as BioT or FTI, has so far proposed been, but has not been confirmed yet. Furthermore, in a recent study carried out on a general population, independently of TT, higher sex hormone‐binding globulin was associated with either subjective or objective androgen deficiency featuresThe significant correlation between TT and BioT is relevant, but both parameters are not associated with ED in the same way. We confirmed here that FTI is not useful.We highlighted the role of hs‐CRP as a possible good predictive marker of ED. The role of hs‐CRP on metabolic syndrome, associated or not with low TT, is well known, particularly in elderly healthy men aged >75 years, but studies targeting men with type 2 diabetes are lackingThe impact of the present finding is very important, as a previous study reported that ED could be the first clinical sign of endothelial dysfunction and a clinical marker of cardiovascular diseases. Finally, our results could change the clinical management of men with diabetes, particularly for the screening of cardiovascular risk factorsBioT or FTI are not good markers to evaluate ED in men aged >55 years with type 2 diabetes. TT lacks specificity in this studied population. However, unexpectedly, we found that hs‐CRP could have a potential role as a predictive marker of ED.The authors declare no conflict of interest.


Narcolepsy is a chronic sleep disorder caused by a loss of hypocretin (hcrt) neurons in the hypothalamus. Cerebrospinal fluid (CSF) hcrt-1 measurement has been well established as a gold standard of narcolepsy diagnosis, although some portions of narcoleptic patients show normal hcrt-1 levels. We aimed to examine peptide degradation of hcrt-1 and its abnormality in the CSF of patients by using high performance liquid chromatography (HPLC) followed by radioimmunoassay (RIA). CSF was collected from healthy controls, narcoleptic patients of type 1 with hcrt-1 deficiency, type 1 with normal hcrt-1 level, and type 2 with normal hcrt-1 level. We found that the majority of hcrt-1 immunoreactivity in extracted CSF was derived from unauthentic hcrt-1 peaks, which are predicted to be inactive metabolites, and the intact hcrt-1 peptide was less than 10% of the gross amount, suggesting that the regular RIA for CSF hcrt-1 measures largely reflect the unauthentic hcrt-1-related metabolites rather than the intact one. As expected, all hcrt-1-related peaks were abolished in type 1 with hcrt-1 deficiency. Importantly, we also found that the sum of the authentic hcrt-1 peptide (peaks 3 and 4) significantly decreased in non-deficient type 1 and tended to decrease in type 2 narcoleptic patients although the levels with the regular RIA in non-extracted CSF was equivalent to healthy controls. Immunoreactivity with unauthentic hcrt-1 metabolites may masks the possible decline in authentic hcrt-1 level caused by the partial loss of hcrt neurons. Our findings may provide new insights into the degradation of the hcrt-1 peptide and the pathophysiology of narcolepsy. Since reduced number of hcrt cells are observed in narcolepsy with cataplexy patients and a mutation in hcrt-related genes is extremely rare in human cases3, CSF hcrt-1 measurement has been well established as a gold standard of diagnosis of narcolepsy by the ICSD-25. However, it has been reported that 10% of narcolepsy with cataplexy and 80–90% of narcolepsy without cataplexy cases have normal CSF hcrt-1 levels6. In addition, normal or moderate hcrt-1 level is one of the current diagnostic criteria for type 2 narcolepsy. However, mechanisms underlying normal hcrt-1 level in narcolepsy are largely unknown and studies focusing on hcrt-1 non-deficient type 1 patients are very limited. Therefore, its clinical features are heterogeneous and the discrepancy between normal hcrt-1 level and clinical symptoms could make the differential diagnosis complex.Narcolepsy is a chronic sleep disorder characterized by excessive daytime sleepiness, cataplexy, sleep paralysis, and hypnagogic hallucinations. The hypocretin (hcrt)/orexin system plays pivotal roles in the etiology of narcolepsy8. The peptide sequence and structure are highly conserved across mammalian species. On the other hand, hcrt-2 is a 28-amino acid with C-terminal amidation. Unlike hcrt-1, its N-terminal half does not include disulfide bonds and thus forms a linear peptide, although its C-terminal half is very similar to that of hcrt-1. Both of the two peptides are exclusively produced in the lateral hypothalamic area. Hcrt neurons widely project to multiple areas throughout the brain except the cerebellum11. Among them, dense projection is observed in the serotonergic raphe nuclei, histaminergic tuberomammillary nucleus, and noradrenergic locus coeruleus where hcrt-1 receptor and/or hcrt-2 receptor are expressed, suggesting that hcrts and their receptors are likely to regulate not only the stability of wake and sleep states but more broadly of brain functions such as feeding, emotion, and reward.Hcrt-1 and -2 are derived from a single precursor polypeptide, prepro-hypocretin, through proteolytic cleavage. Hcrt-1 is a 33-amino acid peptide with a N-terminal pyroglutamyl residue and C-terminal amidation. Hcrt-1 possesses two intrachain disulfide bonds that consist of four cysteine residues (Cys6-Cys12 and Cys7-Cys14) in its N-terminal, and these bonds make a rigid turn conformation14, low rates (0–40%) of hcrt-1 deficiency20, and inconsistency in multiple sleep latency test results21. In addition, nonspecific symptoms further make the diagnosis of narcolepsy without cataplexy challenging22. A postmortem study reported that a narcolepsy without cataplexy patient showed a partial loss of hcrt cells in the posterior hypothalamus, accompanied by prominent gliosis, but not in the anterior hypothalamus23. However, this observation was reported in a single case, and partial hcrt cell loss needs to be further confirmed in more patients. Additionally, evidence linking a focal loss and an absence of cataplexy remains unclear.Studies focusing on narcolepsy without cataplexy have so far been limited due to a more heterogeneous etiology, such as less frequent HLA DQB1*06:02 positivityTo date, little is known about degradation of the hcrt-1 peptide in the CSF. In addition, the regular RIA provides the total value of peptides that are immunoreactive to an anti-hcrt-1 antibody, but it has not been validated if the regular RIA reflects the intact hcrt-1. To address the discrepancy in the CSF hcrt-1 level and nosological aspects of type 1 and 2 narcolepsy, it is important to characterize the degradation of hcrt-1 peptide in type 1 and 2 narcolepsy patients. In this study, we analyzed the HPLC elution pattern and quantified antibody immunoreactive fractions in the CSF.All methods were performed in accordance with the relevant guidelines and regulations. Informed consent in accordance with Stanford guidelines was obtained from all subjects. The research protocol was approved by the Stanford Institutional Review Board on Medical Human Subjects.25. All narcoleptics had been diagnosed based on the clinical symptoms and CSF hcrt-1 level according to ICSD-2 and were regrouped in this study according to ICSD-3 as follows: type 1, narcolepsy-cataplexy with hcrt-1 deficiency (n = 6); non-deficient type 1, narcolepsy-cataplexy with normal levels of hcrt-1 (n = 12); non-deficient type 2, narcolepsy without cataplexy with normal levels of hcrt-1 (n = 5). All control subjects had no clinical sleep abnormalities. CSF samples have been collected by lumber puncture between the years of 1995 and 1997 (between 9 am and 7 pm) from patients and healthy subjects for narcolepsy diagnosis and research purposes, and have been kept frozen at −80 °C. Demographics of samples used are summarized in Table CSF samples from 23 narcoleptics and 10 healthy control subjects were selected from the database at the Stanford University Center for Narcolepsy as previously described125I labeled Orexin A isotope  was separated using the same protocol.All CSF samples have gone through freeze-thaw cycles of less than 3 times before the experiment. CSF samples were centrifuged at 10,000 g for 10 minutes and the supernatant was used for analysis. One ml of CSF samples from patients of type 1, non-deficient type 1, non-deficient type 2, and healthy control subjects were separated by HPLC  at a flow rate of 1 ml/min and linear gradient of 10–60% acetonitrile/0.1% trifluroacetic acid over 40 minutes. Samples were separated by μBondapak C18 column 3.9 × 300 mm, 10 μM 125 A, . Fractions were collected every minute over 40 minutes and were dried up using a vacuum centrifuge system . Samples were stored at −20 °C until use. In order to identify the authentic hcrt-1 peptide, 125I labeled Orexin A isotope according to the manufacture’s instruction. The in-house polyclonal anti-hcrt-1 antibody was raised in rabbits by injecting a full-length hcrt-1 peptide. The IC90 of the RIA was 0.5 pg/ml, which about 20 times more sensitive than the commercially available antibody by Phoenix. A high sensitive assay is more suitable for the purpose of the study and we therefore used our in-house antibody for the RIA. The antibody did not cross-react with hcrt-2 and other related peptides such as the following: growth hormone releasing factor, glucagon, vasoactive intestine peptide, and secretin that belong to the secretin family peptides, bombesin, gastrin releasing peptide, and neuromedin C and B that belong to the bombesin family peptides (unpublished data). The intra- and inter-assay variations were determined by measuring 5 triplicated samples on 3 different days . Intra-assay coefficient of variation was between 3.5 and 6.1%, while inter-assay coefficient of variation was between 2.5 and 6.4%. We also confirmed that the antibody could successfully detect hcrt-1 neurons in rats, zebra fish, dogs, and humans (unpublished data). For the measurement of hcrt-1 immunoreactive peaks in the CSF, fractioned samples were resuspended in 250 µl of deionized water, and 100 µl was used in duplicates for the assay. Radioactivity of each fraction was quantified with a gamma counter . All values are expressed as mean ± SEM. All procedures including human CSF and radioactive material handling were carried out in accordance with guidelines of Stanford Environmental Health and Safety.Hcrt-1 concentration in the CSF with and without extraction was measured using an in-house hcrt-1 antibody and The mean values of hcrt-1 of the 1-minute fractions were plotted as an elution pattern. The area under curve of each peak was calculated and compared among groups using a Kruskal-Wallis test with post-hoc testing using Steel test (JMP).125I-labeled hcrt-1 was fractionalized and measured by RIA. Of the 3 peaks at 3–5, 18–20, and 20–23 minutes, the small peak at 18–20 minutes and the following major peak at 20–23 minutes likely correspond to the authentic hcrt-1. A synthetic hcrt-1 peptide (160 pg of the standard peptide included in a RIA kit) was also separated using HPLC, and a major peak was observed at 18–23 minutes . Four distinguishable peaks  were observed in the CSF from healthy subjects, non-deficient type 1, and non-deficient type 2 . On the other hand, non-deficient type 2 failed to reach the significance  because 2 out of 5 samples showed equivalent values to healthy control. Interestingly, the hcrt-1 concentration in non-extracted CSF among these groups was equivalent to the healthy control group .In order to determine the retention time of the authentic hcrt-1 peak, Since RIA using a polyclonal antibody from Phoenix is widely used for clinical diagnosis test for narcolepsy, we also characterized Phoenix and our in-house antibodies: hcrt-1 levels in extracted CSF from a healthy subject and immunohistochemistry in a mouse brain were compared  patients have a partial loss of hcrt cells, with maximal cell loss in the posterior hypothalamus37. We also cannot exclude the possibility of circadian modulation in degradation because little information is available concerning degradation process of hcrts. Further studies are needed to address these issues and to determine if the HPLC peak pattern we reported here is generally observed in narcoleptic patients with normal hcrt-1 level.Since the RIA measure gives relative value and is methodologically not superior in quantification of small differences, it would be of interest to measure the hcrt-1 level in non-deficient type 1 and type 2 narcolepsy by more specific quantitative methods. It also raises the possibility that hcrt-1 peptide decay might be accelerated in non-deficient cases, yet not much is known about the degradation process of hcrts. Some factors that may influence the degradation deserve consideration. In this study, we used CSF samples that were stored at −80 °C for a long period. Advanced decay during storage could be ruled out because freshly collected CSF also showed a similar peak pattern and the authentic hcrt-1 level was already low. It is difficult to speculate on the effect of medication on hcrt degradation from only 1 or 2 non-medicated patients in each group. It has been reported that CSF hcrt-1 level shows seasonal fluctuation in humans and rodents and with a clear diurnal rhythmSupplementary information


Meningeal carcinomatosis (MC) is the most severe form of brain metastasis and causes significant morbidity and mortality. Currently, the diagnosis of MC is routinely confirmed on the basis of clinical manifestation, positive cerebrospinal fluid (CSF) cytology, and/or neuroimaging features. However, negative rate of CSF cytology and neuroimaging findings often result in a failure to diagnose MC from the patients who actually have the disease. Here we evaluate the CSF circulating tumor DNA (ctDNA) in the diagnosis of MC.A total of 35 CSF samples were collected from 35 patients with MC for CSF cytology examination, CSF ctDNA extraction and cancer-associated gene mutations detection by next-generation sequencing (NGS) at the same time.p = 22/25) and 63%  (p = 22/35) compared to those of CSF cytology and CSF ctDNA, respectively. The sensitivity of the CSF cytology was 71%  (n = 25/35) compared to that of CSF ctDNA.The most frequent primary tumor in this study was lung cancer , followed by gastric cancer , breast cancer , prostatic cancer , parotid gland carcinoma  and lymphoma  while no primary tumor could be found in the remaining 2 patients in spite of using various inspection methods. Twenty-five CSF samples  were found neoplastic cells in CSF cytology examination while all of the 35 CSF samples  were revealed having detectable ctDNA in which cancer-associated gene mutations were detected. All of 35 patients with MC in the study underwent contrast-enhanced brain MRI and/or CT and 22 neuroimaging features  were consistent with MC. The sensitivity of the neuroimaging was 88%  (This study suggests a higher sensitivity of CSF ctDNA than those of CSF cytology and neuroimaging findings. We find cancer-associated gene mutations in ctDNA from CSF of patients with MC at 100% of our cohort, and utilizing CSF ctDNA as liquid biopsy technology based on the detection of cancer-associated gene mutations may give additional information to diagnose MC with negative CSF cytology and/or negative neuroimaging findings. Meningeal carcinomatosis (MC), also called neoplastic meningitis (NM) is the most severe form of brain metastasis and causes significant morbidity and mortality . The earCurrently, the diagnosis of MC is routinely confirmed on the basis of clinical signs and symptoms, cerebrospinal fluid (CSF) cytology, and/or neuroimaging (contrast-enhanced brain MRI and/or CT) findings . The preCirculating tumor DNA (ctDNA) consists of short, double stranded DNA fragments that are released by tumors . The ctDWritten informed consent has been obtained from all patients or their legal surrogates. The study protocol has been approved by the Ethics Committee of the Second Hospital of Hebei Medical University, Hebei, China. The patients with MC were enrolled from Department of Neurology in the Second Hospital of Hebei Medical University. In total, 35 patients with MC who underwent lumbar puncture for CSF cytology examination, CSF ctDNA extraction and cancer-associated gene mutations detection by next-generation sequencing (NGS) at the same time and underwent contrast-enhanced brain MRI and/or CT were enrolled in this study between October 2014 and September 2017. The diagnosis of MC had been established by clinical signs and symptoms in addition to positive CSF cytology and/or neuroimaging findings (contrast-enhanced brain MRI or CT) consistent with MC. The clinical signs and symptoms included headache, nausea and vomiting, convulsion, lower back pain, cranial nerve paralysis, paresthesia, gait disturbances, vertigo and defects in mental functioning. The positive CSF cytology result was defined by the morphology of neoplastic cells such as different size with irregular-shape, big nucleus with malignant signs such as lobulated state and malformed buds, increasing chromatin with basophilic coarse particles, mitotic activity with aberrant mitosis, obvious and polymorphic nucleolus accounting for the majority of chromatin and thickening nuclear membrane with saw-tooth-shaped and wear edge. The positive neuroimaging finding was defined as the presence of leptomeningeal enhancement.Cerebrospinal fluid samples were collected from each patient. CSF samples in EDTA tubes were centrifuged for 5 min at 1000 g. The pellet was stored at − 20 °C, while the supernatant was centrifuged at 10,000 g for another 30 min . The supPreparation of the Ion Proton library and DNA sequencing were performed as described in our previous publications –22. For We used the SV-OCP143-ctDNA panel , which is capable of detecting somatic mutations from plasma or tissue samples on 143 cancer-related genes. Since the ctDNA in CSF is comprised of short DNA fragments, amplicons in the panel are specially designed for efficient amplification of ctDNA. For CSF samples, the total read count was more than 25 million to ensure the average base coverage depth over 10,000 x. Additionally, the average uniformity of base coverage was 95.5%. These strict quality control criteria ensured the reliability of sequencing.p value<0.01. The second-step utilized the Integrative Genomics Viewer (IGV) software (http//www.broadinstitute.org/igv) or Samtools software (http://samtools.sourceforge.net) to eliminate possible DNA strand-specific errors after visual examination of called mutations.Initial data from the sequencing runs were processed with the Ion Proton platform-specific pipeline software Torrent Suite v5.0 including generating sequencing reads, trim adapter sequences, filtering and removing poor signal-profile reads as described in our previous publications –22. InitThe clinical characteristics of 35 patients with MC were summarized in Table The sensitivity of all methods analyzed was presented in Table p = 22/25) and 63%  (p = 22/35) compared to those of CSF cytology and CSF ctDNA, respectively. The sensitivity of the CSF cytology was 71%  (n = 25/35) compared to that of CSF ctDNA. The specificity of CSF ctDNA in all cases was 100%. These data suggest a higher sensitivity of CSF ctDNA than those of CSF cytology and neuroimaging findings.The sensitivity of the neuroimaging was 88%  (Nine cases were CSF cytology negative but neuroimaging positive while CSF ctDNA with cancer-associated gene mutations could be detected in them. Interestingly, neoplastic cells were found in CSF of patient N33 after 1 year from the first lumber puncture (Fig. The comparisons between primary tumors and MC CSF samples were available only in 6 MC patients with lung adenocarcinoma Table . We collIn the present study, we compared the sensitivity of three methods including CSF cytology, neuroimaging findings and CSF ctDNA in the diagnosis of MC. The study revealed that CSF ctDNA had a higher sensitivity than the CSF cytology and neuroimaging findings. Most studies of ctDNA published to date have demonstrated the importance of plasma ctDNA as a liquid biopsy medium for various kinds of cancer given that benign tumors and non-neoplastic conditions do not generally give rise to ctDNA . SimilarCurrently, the presence of neoplastic cells in the CSF is the most useful criteria to confirm the diagnosis of MC and CSF cytology remains the gold diagnostic standard although the false negative rate of CSF cytology is still high. In our study, we found the sensitivity of CSF ctDNA is higher than that of CSF cytology. Previous study  had presIn cancer patients, ctDNA are thought to be released in plasma as a result of tumor cells apoptosis and/or necrosis , 25. ctDElena et al.  used nexAs per the analysis for concordance of EGFR activating mutations between primary lung adenocarcinomas and MC, on the basis of patients’ CSF samples, the results showed that, EGFR activating mutations in CSF samples were consistent with those in primary adenocarcinomas. The showing of T790 M in two CSF samples, is possibly attributed to the fact that both two CSF samples were sequentially collected during the process of TKI therapy.Our study find DNA mutation in CSF of patients with MC at 100% of our cohort, and it may give additional information to diagnose MC with negative CSF cytology. Furthermore, the type of tumor-associated gene mutations can provide many clues to the primary tumor type especially for patients whose primary tumor wouldn’t be found in spite of using various inspection methods. Additionally, looking for and knowing the primary tumor is the rather important diagnostic dependency.However, our current study does have some limitations; for example, the cohort has had relative small numbers of patients and we used a smaller 143 gene panel. Though it was the most comprehensive gene panel at that time, including hot-spot mutation, copy number variants, gene fusion, and gene therapy information, it could not detect all possible aberrations in DNA. Thus, future study with larger size of samples from other departments and multiple institutions and using larger gene panel could help us to solve these issues.This study suggests a higher sensitivity of CSF ctDNA than those of CSF cytology and neuroimaging findings. We find cancer-associated gene mutations in ctDNA from CSF of patients with MC at 100% of our cohort, and utilizing CSF ctDNA as liquid biopsy technology based on the detection of cancer-associated gene mutations may give additional information to diagnose MC with negative CSF cytology and/or negative neuroimaging findings. At the same time, cancer-associated gene mutations provide a reliable theory for MC targeted therapy.


RBP4, retinol and the RBP4/retinol ratio were not different between T2DM and non-diabetic subjects , and were unrelated to body mass index. Notably, RBP4 and retinol were elevated in subjects with metabolic syndrome (p < 0.05), which was attributable to an association with elevated triglycerides (p = 0.013). Large VLDL, total LDL and small LDL were increased in T2DM subjects (p = 0.035 to 0.003). Taking all subjects together, RBP4 correlated with total cholesterol, non-HDL cholesterol, LDL cholesterol, triglycerides and apolipoprotein B in univariate analysis (p < 0.001 for each). Age-, sex- and diabetes status-adjusted multivariable linear regression analysis revealed that RBP4 was independently associated with large VLDL  and small LDL particles . Its relationship with large VLDL remained after further adjustment for retinol. RBP4 did not co-elute with VLDL nor LDL particles in FPLC analyses. Conclusions: Plasma RBP4 levels are related to but do not physically interact with large VLDL and small LDL particles. Elevated RBP4 may contribute to a proatherogenic plasma lipoprotein profile.Background: Retinol binding protein 4 (RBP4) carries retinol in plasma, but is also considered an adipokine, as it is implicated in insulin resistance in mice. Plasma RBP4 correlates with total cholesterol, low density lipoprotein (LDL)-cholesterol and triglycerides, and may confer increased cardiovascular risk. However, controversy exists about circulating RPB4 levels in type 2 diabetes mellitus (T2DM) and obesity. Here, we analyzed the relationships of RBP4 and retinol with lipoprotein subfractions in subjects with and without T2DM. Methods: Fasting plasma RBP4 (enzyme-linked immunosorbent assay) and retinol (high performance liquid chromatography) were assayed in 41 T2DM subjects and 37 non-diabetic subjects. Lipoprotein subfractions (NMR spectroscopy) were measured in 36 T2DM subjects and 27 non-diabetic subjects. Physical interaction of RBP4 with lipoproteins was assessed by fast protein liquid chromatography (FPLC). Results: Plasma RBP4 and retinol were strongly correlated (r = 0.881,  The incidence of the metabolic syndrome (MetS) and Type 2 diabetes mellitus (T2DM) has rapidly increased in the past few years . Both MeRetinol-binding protein 4 (RBP4) is produced by the liver and adipose tissue and transports retinol (vitamin A) via the circulation to peripheral tissues. However, RBP4 is also considered to be an adipokine, as it has been implicated in the pathogenesis of insulin resistance . EnhanceIn contrast to the equivocally reported relationships of circulating RBP4 with obesity, dysglycemia and insulin resistance, positive relationships of RBP4 with total cholesterol, low density lipoprotein (LDL) cholesterol and triglycerides have repeatedly and almost invariably been demonstrated ,15,16,17Therefore, the present study aimed to determine whether plasma levels of RBP4 and/or retinol relate to VLDL and LDL subfractions in adults with and without T2DM.2 and/or proteinuria), abnormal liver function tests (transaminases > 3 times the upper reference limit) or thyroid dysfunction (thyroid stimulating hormone > 10 or < 0.40 mU/L or use of thyroid function influencing medication), as well as current smoking and use of lipid lowering drugs was also an exclusion criterion. Blood pressure was measured after 15 min of rest in the left arm in sitting position using a sphygmomanometer. Body mass index (BMI in kg/m2) was calculated as weight divided by height squared. Waist circumference was measured on bare skin between the 10th rib and the iliac crest. The participants were studied after an overnight fast. Metabolic syndrome (MetS) was defined according to NCEP-ATP III criteria [The study protocol was approved by the medical ethics committee of the University Medical Center Groningen. All participants provided written informed consent and were aged > 18 years. They were recruited by advertisement in local newspapers. T2DM was diagnosed by primary care physicians based on fasting plasma glucose ≥ 7.0 mmol/L and/or a non-fasting plasma glucose ≥ 11.1 mmol/L. Diabetic patients using metformin, sulfonylurea and antihypertensives were included. Insulin use was an exclusion criterion. A positive history of cardiovascular disease, chronic kidney disease . Plasma total cholesterol and triglycerides were measured by routine enzymatic methods . HDL cholesterol was assayed by a homogeneous enzymatic colorimetric test . Non-HDL cholesterol was calculated as the difference between total cholesterol and HDL cholesterol. LDL cholesterol was calculated by the Friedewald formula if triglycerides were <4.5 mmol/L. Apolipoprotein A-I (apoA-I) and apolipoprotein B (apoB) were measured by immunoturbidimetry . HbA1c was measured by high-performance liquid chromatography .Plasma RBP4 was assayed by enzyme-linked immunosorbent assay . In brief, 96-well plates precoated with anti-human RBP4 monoclonal antibody were stored at 4 °C. After adding 200 μL of 2 vials  of a buffered protein solution to each well, 20 μL of sample material was added to each well and left for 1 h at room temperature on a horizontal orbital microplate shaker (0.12” orbit) set at 500 rpm. This procedure was followed by aspiration and washing of each well with 400 μL buffered surfactant, repeating the process three times for a total of four washes. 200 μL of monoclonal antibody specific for human RBP4 conjugated to horseradish peroxidase were added to each well and incubated for 1 h at room temperature under gentle agitation. After repeating aspiration/wash as described, 200 μL of Substrate Solution consisting of stabilized hydrogen peroxide and stabilized chromogen (tetramethylbenzidine) were added to each well and incubated for 30 min at room temperature under protection from light. The reaction was then stopped by adding 50 μL of 2 N sulfuric acid; the absorbance was measured at 450 nm, 540 nm and 570 nm.Plasma retinol levels were analyzed by reverse phase high performance liquid chromatography (HPLC) as described . BrieflyLipoprotein separation was performed by fast protein liquid chromatography (FPLC) as described before . One mL Lipoprotein particle profiles were determined by nuclear magnetic resonance (NMR) spectroscopy with the LP3 algorithm  ,31. Verye transformed to achieve approximately normal distributions. Between group differences in variables were determined by T-tests for unpaired data and Chi-square tests where appropriate. Univariate correlations were determined by Pearson correlation coefficients. Multivariable linear regression analyses were carried out to disclose the independent relationships of RBP4, retinol and the RBP4/retinol ratio with either T2DM or MetS. Additionally, multivariable linear regression analyses were performed to disclose the independent associations with RBP4 and retinol with VLDL, LDL and HDL subfraction characteristics, each in separate models. Two-sided p-values < 0.05 were considered statistically significant.IBM SPSS software  was used for data analysis. Continuous variables were expressed in means ± SD or medians (interquartile ranges) in case of not-normally distributed data. HOMA-IR, triglycerides and lipoprotein subfractions were logForty-one (41) subjects with T2DM and 37 subjects without T2DM were included. Clinical characteristics and biochemical data are presented in p = 0.63; retinol, p = 0.25; RBP4/retinol ratio, p = 0,19, respectively; data not shown). In contrast, RBP4 and retinol levels were higher in subjects with MetS vs. subjects without MetS . As a consequence, the RBP4/retinol ratio was not different between subjects with and without MetS . The associations of RBP4 and retinol with the presence of MetS remained close to significance after adjustment for age and sex . Additionally, in age- and sex-adjusted multivariable linear regression analysis, RBP4 was independently associated with elevated triglycerides , but not with the other individual MetS components (p ≥ 0.75 for each). Serum retinol levels were not associated with any of the MetS components . In univariate analysis, RBP4 and retinol were not significantly associated with HOMA-IR . Furthermore, RBP4, retinol and the RBP4/retinol ratio were not significantly different between men and women .As expected, T2DM subjects were classified with MetS more frequently, had higher blood pressure, were more obese and had higher glucose and HbA1c levels and had higher HOMA-IR when compared to non-diabetic controls . In geneLipoprotein subfractions were quantified in 36 T2DM subjects and 27 non-diabetic subjects . T2DM pap < 0.001), as well as in T2DM subjects  and non-diabetic subjects separately   . In all < 0.001) A–C.p < 0.001), small LDL  and large HDL  were correlated with HOMA-IR in all subjects combined. Comparable relationships were found in T2DM and non-diabetic subjects separately (data not shown).With respect to lipoprotein subfractions, we observed positive univariate correlations of RBP4 and retinol with VLDL-P, large and medium VLDL, LDL-P, small LDL and small HDL, but inverse correlations with large HDL A–C. Agaip < 0.001) and retinol , of small LDL with RBP4  and with retinol , and the inverse relationship of large HDL with RBP4  and retinol  remained statistically significant. Remarkably, when retinol was added as an independent variable to the model of RBP4 with VLDL subfractions (p = 0.022). Conversely, when RBP4 was added as independent variable in the model of retinol with VLDL subfractions (p = 0.28).We next performed multivariable linear regression analysis to disclose the extent to which RBP4 and retinol were independently related to VLDL, LDL and HDL subfractions . In age-ractions , model Aractions , model BGiven the correlation of plasma RBP4 levels with certain subfractions, we also assessed whether RBP4 may actually physically interact with such lipoprotein particles. Lipoprotein particles, as well as soluble proteins, were separated by FPLC from freshly-obtained (non-frozen) plasma of non-diabetic individuals and fractions were analyzed for cholesterol, triglycerides, protein and RBP4 concentrations. A representative FPLC profile is shown in This study confirms and extends previous findings ,13,16,17In our study, plasma RBP4 levels were not elevated in T2DM subjects, in agreement with earlier studies ,13, but RBP4 levels were predominantly associated with large VLDL particles, as well as with small LDL particles, the latter finding being consistent with earlier observations using conventional methods for lipoprotein subfraction quantification . Given tWhile the current study was primarily focused on RBP4, our findings with respect to retinol levels are also relevant. Retinol is the circulating form of vitamin A that is locally (in tissues) converted to retinoic acids  that activate transcription factors (Retinoic Acid Receptor (RAR) and Retinoic X Receptor (RXR)) ,36. RetiIn view of the accumulating though sometimes controversial epidemiological evidence indicating that RBP4 is associated with incident CVD ,26,39,40Several methodological issues of the present study need consideration. Diabetic subjects using insulin and lipid lowering were not allowed to participate, in order to obviate confounding due to effects of insulin and lipid-lowering drugs on glucose and lipid metabolism. As a consequence, T2DM subjects with rather mild hyperglycemia and dyslipidemia were preferentially included, which could to some extent mask relationships of RBP4 with dysglycemia and could underestimate relationships of RBP4 with (apo)lipoproteins and lipoprotein subfractions. Furthermore, we excluded subjects with impaired glomerular filtration rate. This was done to minimize confounding with respect to circulating RBP4 due to chronic kidney disease in view of the inverse relationship of RBP4 with glomerular filtration rate . ObviousSerum RBP4 levels are related to large VLDL and small LDL particles, but in the absence of a physical interaction between RBP4 and the lipoprotein particles. Higher RBP4 may be part of a proatherogenic plasma lipoprotein profile. However, it seems unlikely that alterations in circulating RBP4 per se contribute significantly to the predominance of large VLDL and small LDL in the diabetic state.