There is a recent debate on the "transplantability" of ECD (Expanded Criteria Donors) kidneys and the selection criteria used to allocate them to single or double transplantation. Remuzzi et al. have defined a protocol incorporating pre-transplant donor biopsy to guide the use of older donor organs. They allocated organs as single or double transplants on the basis of histological findings. We aim to show the pros and cons of the only histological evaluation in the allocation of ECD kidneys, to compare the different experiences in United States and Europe and thus to discuss whether this tool should be used alone or included in a comprehensive clinical and histopathological evaluation.In the United States many Authors stated that the biopsy actually increases the percentage of kidney discarded and they raised questions about the importance of the biopsy in evaluating ECD kidneys for transplantation. On the other hand, the experiences of the majority of european transplant centers showed that allocating kidneys as single or dual transplant based on biopsy findings may achieve good graft and patient outcomes.Moreover, the experience of some centers as ours showed that kidneys allocated as DKT  on the basis of Remuzzi’s score could have been suitable for single transplantation suggesting the need of an adjustment of the Remuzzi Score System. Many Authors, who are in favor of histological evaluation, actually believe that a comprehensive clinical and histopathological assessment before transplantation remains necessary.We lack precise national- or international-based selection criteria to guide clinicians. An adjustment of the Remuzzi Score System could be taken into consideration such as narrowing the indication for DKT from those ECD kidneys with higher scores and including the histological evaluation in a multifactor score. The disparity between supply of deceased donors and demand has increased in the last decades and has led to the increased use of organs from marginal donors, mostly in form of expanded criteria donor (ECD) kidneys–7. ECD iBecause of the decreased chance of survival of renal grafts from ECDs, the transplantation of two ECD kidneys into a single recipient  has been introduced. It is considered when a single transplant exposes the recipient to the drawbacks of a limited nephron mass supply. This strategy has provided good outcomes in the experience of some transplant center–19.To solve the problem of maximizing the use of ECD kidneys and choosing which ECD kidneys should be used for single or double use, it has become important to develop tools that permit an accurate evaluation of their "transplantability". Remuzzi et al. have defined a protocol incorporating pre-transplant donor biopsy to guide the use of older donor organs. They allocated organs as single or double transplants on the basis of histological findings. They reported excellent outcomes of the Dual Kidney Transplant Group at 5 years of follow-up. HoweverWe aim to show in this review the pros and cons of the only histological evaluation in the allocation of ECD kidneys, to compare the different experiences in United States (US) and Europe and thus to discuss whether this tool should be used alone or included in a comprehensive clinical and histopathological evaluation.In European countries, lack of elderly recipients on the waiting list as well as macroscopic or microscopic kidney abnormalities were the main causes for organ discard. On the other hand, biopsy findings and machine perfusion have revealed major reasons to discard ECD in the United States.US transplant centers continue to discard an high percentage of deceased donor kidneys that have been procured for transplantation. Between October 1999 and June 2005, more than 9000 donated kidneys were determined to be unsuitable for transplantation, including 41% (5139 of 12536) of the expanded criteria donor (ECD) kidneys procured, according to an analysis from the Scientific Registry of Transplant Recipients in this issue. Sung etMoreover, the Authors considered that there may be important selection biases at work that cannot be detected in Registry analyses and that the degree of glomerulosclerosis is the only biopsy result that can be evaluated from the forms that are submitted. Glomerulosclerosis does not appear to be accurate histologic indicator risk of subsequent renal function. ProbablTo develop a tool to reduce the discard rate of marginal kidneys in US, the United Network for Organ Sharing (UNOS) Kidney Transplantation Committee has approved a new allocation system on June 25, 2013. This new system uses the Kidney Donor Risk Index (KDRI) to steamline allocation of kidneys which otherwise would have been discarded. The KDRI combines a variety of donor factors to summarize the risk of graft failure after kidney transplant into a single number. The following donor characteristics are used to calculate the KDRI: age, height, weight, ethnicity, history of hypertension, history of diabetes, cause of death, serum creatinine, Hepatitis C Virus (HCV) status, Donation after Circulatory Death (DCD) status. The KDPI (Kidney Donor Profile Index) is a remapping of the KDRI onto a cumulative percentage scale, such that a donor with a KDPI of 80% has higher expected risk of graft failure than 80% of all kidney donors recovered last year. Clinical applicability of a continuous variable has limitations in a binary decision-making process like acceptance or rejection of a kidney (if there is not a cutoff). Thus one should have great caution in utilizing KDPI to predict a specific patient's suitability for that organ or for discriminating kidneys that are suitable for transplantation, 24.With the goal to find a tool for identification of ECD kidneys that can be accepted for tranplantation, Gandolfini et al. analyzedIn order to reduce discard rate of marginal kidneys a potential option is to allocate them as a DKT. In Europe DKT proved to be a successful strategy for compensating the declining number of cadaver donor kidney transplants. On the other hand DKT is not a part of the allocation system in US and comprises less than 2% of the deceased donor transplants performed. The criteria proposed by UNOS recommend consideration of DKT if any two of the following criteria exist: donor age greater than 60 years; estimated donor creatinine clearance less than 65 ml/min, rising serum creatinine  at time of retrieval; history of medical disease in donor (defined as either longstanding hypertension or diabetes mellitus); adverse donor kidney histology, which is defined as moderate to severe glomerulosclerosis (greater than 15% and less than 50%). Potential factors associated with low utilization of DKT in US are higher surgical risk, longer ischemic time and the uncertainty in determining which organs are suitable for single versus dual use.According to the prospective cohort study of the Dual Kidney Transplant Group in Italy, the pretransplant biopsy is an important tool to optimize the allocation and utilization of ECD kidneys.Nevertheless the experience of some centers as ours showed that kidneys allocated as DKT on the basis of Remuzzi’s score could have been suitable for single transplantation. Cruzado et al. demonstrOther studies showed the need of an adjustment of the criteria proposed by Remuzzi et al. For instance, Fernandez-Lorente et al. have evaThere are several methods reported in literature to assess the quality of ECD kidneys and so to allocate them to single or dual transplant. Transplantation centers may make this decision based on a single criterion or on a comprehensive clinical and histological assessment. In 2006, Remuzzi et al. reported the long-term graft survival of 62 patients who received a transplant  from donors over 60 years of age. In this study, marginal kidneys with no macroscopic abnormalities were allocated on the basis of the only histological evaluation of the grafts before transplantation: histological changes in the vessels, glomeruli, tubules, and connective tissue in biopsy specimens received a score from 0 (no changes) to 3 (severe changes). When both donor kidneys had a total score of 0 – 3, each kidney was used for a SKT. When the total score was 4 – 6, the two kidneys were transplanted in the same recipient (DKT). Kidneys with a total score ≥ 7 were not utilized. 3 years after transplantation, recipients of SKT and DKT organs from donors over 60 years of age that had undergone preimplantation biopsy had a mean graft survival rate > 90%, which was similar to that observed in a matched cohort of recipients of SKT renal grafts from donors < 60 years of age. Moreover, the survival rate of grafts from donors > 60 years of age that had undergone preimplantation biopsy was significantly higher than that observed in a matched cohort of recipients of organs from donors > 60 years of age that had not undergone preimplantation biopsy (93% versus 72%). Other studies have tried to define the predictive value of pretransplant biopsy with opposed results. For instance, B. Ekser et al., 31 use However, other scores have been proposed to allocate organs, such as glomerular filtration rate (GFR) alone, or GFR in combination with clinical, epidemiological, or histological criteria, but it has not defined a standardized score to address ECD allocation yet–46. MoreThe transplant community should develop strategies to maximize the yield of the existing donor pool. The use of kidneys from ECD may permit more transplantations. For ECD kidneys unsuitable for single use, DKT may be possible. In many centers kidneys from ECD are histologically evaluated before implantation to decide the allocation for single or double use, but other scores have been proposed to allocate organs and there is not yet a standardized score to address ECD allocation. According to some studies, only kidneys with Remuzzi’s score of 5 and 6 should be used for DKT. Therefore, an adjustment of the Remuzzi Score System could be taken into consideration such as narrowing the indication for DKT from those ECD kidneys with higher scores and including the histological evaluation in a multifactor score.CG, specializzando in Chirurgia Generale, Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, ItalyVD, dirigente medico, Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, ItalyMD, ricercatore di Anatomia Patologica, Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, ItalySC, specializzando in Anatomia Patologica, Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, ItalyAS, dirigente medico, Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, ItalyLP, dirigente medico, Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, ItalyAR, professore ordinario di Chirurgia Generale, Dipartimento di Scienze Biomediche Avanzate, Università Federico II, Naples, Italy


To confirm if a real inner descend of testis occurs, correlating the testicular position with fetal parameters and analyzing the position of the testes relative to the internal ring. Twenty nine human fetuses between 13 and 23 weeks post conception (WPC) were studied. The fetuses were carefully dissected with the aid of a stereoscopic lens with 16/25X magnification and testicular position observed. With the aid of a digital pachymeter the distance between the lower pole of the kidney and the upper extremity of the testis (DK-T) was measured to show the position of the testis. During the dissection we also indicated the position of the testes relative to the internal ring. Means were statistically compared using simple linear regression and the paired T-test. The 58 testes had abdominal position. The DK-T in the right side measured between 0.17 and 1.82cm (mean=0.79cm) and in the left side it was between 0.12 and 1.84cm (mean=0.87cm), without statistically differences (p=0.0557). The linear regression analysis indicated that DK-T in both sides correlated significantly and positively with fetal age. All fetuses with more than 20 WPC, heavier than 350g and with CRL over 22cm had a greater distance than the average DK-T. We xobserved that the 58 testis remains adjacent to the internal ring throughout the period studied. The testes remains adjacent to the internal ring throughout the period studied, indicating that there is no real trans-abdominal testicular descent during the second gestational trimester. Some on (WPC) -3.Various factors have been proposed as causing testicular descent in humans, including the increase in the intra-abdominal pressure , 6, the cess starts at about the 24thweek post-conception, while Heyns , total length of the fetus and the position of the testes relative to the internal ring.This study received institutional review committee approval and was carried out in accordance with the ethical standards of the hospital’s institutional committee on human experimentation.During the period from January 2014 through March 2015, 29 male human fetuses (58 testes) ranging in age from 13 to 23 weeks post-conception (WPC) were studied. The fetuses were macroscopically well preserved, showed no signs of malformations and came to our laboratory as a donation of the obstetric section of our hospital and the demise was hypoxia. The gestational age of the fetuses was determined in WPC, according to the foot-length criterion. This criterion is currently considered the most acceptable parameter to calculate gestational age  Abdominal, when the testis was proximal to the internal ring; b) Inguinal, when the testis was found between the internal and external inguinal rings; and c) Scrotal, when the testis had passed beyond the external inguinal ring and was inside the scrotum.With the aid of a digital pachymeter, the distance between the lower pole of the kidney and the upper pole of the testis (DK-T) was measured to show the descent of the testis during migration. The same observer made these measurements . During Mean values of the distance between the lower pole of the kidney and the upper extremity of the testis for each side were statistically compared using the unpaired T-test. We also performed simple linear regression to assess the association between the variables analyzed with fetal age. In addition, the correlation coefficient (r) and the p-value were obtained for each regression analysis, and p≤0.05 was considered to indicate statistical significance. The Graph pad Prism 5.0 software was used.The fetuses weighed between 30 and 793.4g; had crown-rump length between 7 and 24.5cm and had total length between 10 and 34.5cm. The 58 testes all had abdominal position. The DK-T in the right side measured between 0.17 and 1.82cm  and in the left side between 0.12 and 1.84cm , without statistically significant difference (p=0.0557). Table-1 reports the fetal parameters and the measurements of DK-T in the right and in the left side of all fetuses studied.Considering the average of DK-T in the right and in the left side, all fetuses with more than 20 WPC had a greater distance than the average DK-T value. All fetuses heavier than 350g and with CRL greater than 22cm had the high or very near average DK-T values.2) and the p-values of all fetal parameters studied.The linear regression analysis indicated that DK-T in the right and left sides correlated significantly and positively with fetal age and weight, during the fetal period studied (13 to 23 WPC). When comparing the DK-T with TL and CRL, we observed a weak correlation in the right and in the left side. We observed that the 58 testis remained adjacent to the internal ring throughout the period studied .Previous studies show that in the first phase of testicular descent, the testis descends from the lower pole of the kidney to the pelvic cavity near the bladder neck . In thisth WPC and lasts until the 15th WPC , indicating that there is no real trans-abdominal descent of the testis during the second trimester. Previous studies indicated that the testis has progressive descent until the 15findings , 23. WPC, indWe should mention some limitations of this study: 1) Small sample size–access to human fetuses is limited, so observations of this sample of 29 fetuses may be important although the small number is a weakness, mainly regarding statistical results. 2) Unequal WPC distribution in the period studied–we did not have fetuses with less than 13 WPC and at some ages we had 4 fetuses and at others only 1 or 2 fetuses. Nevertheless, the sample distribution during this important period of testicular migration was adequate in our opinion.The distance between the kidney and the testis in both sides had a strong positive correlation with fetal age and weight in human fetuses. The testes remains adjacent to the internal ring throughout the period studied (13 to 23 WPC), indicating that there is no real trans-abdominal testicular descent during the second trimester.


Plasminogen activation is a ubiquitous source of fibrinolytic and proteolytic activity. Besides its role in prevention of thrombosis, plasminogen is involved in inflammatory reactions in the central nervous system. Plasminogen has been detected in the cerebrospinal fluid (CSF) of patients with inflammatory diseases; however, its origin remains controversial, as the blood–CSF barrier may restrict its diffusion from blood.n = 15), multiple sclerosis (n = 19) and noninflammatory neurological diseases (n = 8).We investigated the origin of plasminogen in CSF using Alexa Fluor 488–labelled rat plasminogen injected into rats with systemic inflammation and blood–CSF barrier dysfunction provoked by lipopolysaccharide (LPS). Near-infrared fluorescence imaging and immunohistochemistry fluorescence microscopy were used to identify plasminogen in brain structures, its concentration and functionality were determined by Western blotting and a chromogenic substrate assay, respectively. In parallel, plasminogen was investigated in CSF from patients with Guillain-Barré syndrome  = 0.66 to 1.59)) as compared to multiple sclerosis (median = 0.3 ng/μl (IQR = 0.16 to 0.61)) and to noninflammatory neurological diseases (median = 0.27 ng/μl (IQR = 0.18 to 0.35)).Our findings demonstrate that plasminogen is transported from circulating blood into the CSF of rats via the choroid plexus during inflammation. Our data suggest that a similar mechanism may explain the high CSF concentrations of plasminogen detected in patients with inflammation-derived CSF barrier impairment.The online version of this article (doi:10.1186/s12974-014-0154-y) contains supplementary material, which is available to authorized users. Pericellular proteolysis in the central nervous system (CNS) is involved in development, regeneration of nervous tissues, neuronal and synaptic plasticity and the inflammatory response [. However, the question has not been settled as to whether plasminogen in CSF originates from circulating blood or is expressed in the CNS, as suggested by the presence of mRNA in mouse brain [Vessel wall fibrinolytic activity and pericellular proteolysis in tissues requires plasminogen binding and transformation into plasmin at the surface of fibrin, cells or the extracellular matrix by either the tissue-type plasminogen activator (tPA) or the urokinase-type plasminogen activator (uPA) . The binammation -5. Pericresponse -9. In huresponse . Trace aresponse , subaracresponse  and multresponse . Howeverse brain ,15 or byse brain .The CNS is protected from the bloodstream by the blood–brain barrier (BBB) and the blood–CSF barrier ,18. The Because diffusion of plasma proteins into the CNS is a selective process, we sought to demonstrate that circulating plasminogen may cross the blood–CSF barrier and can be detected in the CSF. For that purpose, we studied control rats and rats with lipopolysaccharide (LPS)-induced systemic inflammation. Our results show that circulating plasminogen enters the CSF space during blood–CSF barrier dysfunction induced by systemic inflammation, thus suggesting that plasminogen found in the CSF of patients with inflammatory neurological disorders originates from circulating blood.para-nitroaniline (CBS0065) was purchased from Stago . Escherichia coli LPS serotype 0111:B4, trans-4-(aminomethyl)cyclohexane-1-carboxylic acid , amiloride and 4′,6-diamidino-2-phenylindole (DAPI) and Evans blue were obtained from Sigma Chemical Co . Alexa Fluor 488 succinimidyl ester was purchased from Life Technologies . Goat anti-collagen type IV (Col IV) used for immunohistochemical analyses was obtained from Southern Biotech , and donkey anti-goat antibody F(ab′)2 fragments linked to tetramethylrhodamine isothiocyanate were purchased from Jackson ImmunoResearch . Glu-plasminogen, plasmin and peroxidase-conjugated monoclonal antibody against plasminogen kringle 1 (CPL15-PO) were prepared and characterized as described previously [The enhanced chemiluminescence reagent kit was obtained from Bio-Rad Laboratories . The chromogenic substrate selective for plasmin -hydroxyprolylarginine-eviously -21. A raDiagnostic and Statistical Manual of Mental Disorders, Fourth Edition–Text Revision [3) or with blood contamination (erythrocyte concentration >50 cells/mm3) were excluded. The CSF to serum albumin quotient  was calculated and used to evaluate blood–CSF barrier integrity. Qalb > 0.007 was considered a marker of blood–CSF barrier dysfunction [A total of 42 patients with neurological diseases attending the Neurological Clinic of the Careggi University Hospital, Florence, Italy, were admitted in this study. Informed consent was obtained according to the Declaration of Helsinki. The Careggi University Hospital Review Board approved the protocol. Diagnoses were based on clinical, laboratory and magnetic resonance imaging data according to the International Classification of Diseases, Tenth Revision, and the Revision . Patientfunction ,24. The Experiments were performed using male Wistar rats (280 to 350 g) in accordance with the directives of the Council of the European Communities (86/609/EEC) and the French Agriculture and Forestry Ministry for handling animals (decree 87-848).in vivo in the rat model of inflammation, we used fluorescence-labelled native plasminogen purified from rat plasma. Blood was obtained by cardiac puncture of anaesthetized animals and collected in 0.129 M sodium citrate. The plasma was separated from blood by centrifugation, and plasminogen was isolated by lysine affinity and sieving chromatography as described previously [r = 92,000 Da) similar to that of human plasminogen and was labelled with Alexa Fluor 488 dye (A488-Pg) according to the instructions of the manufacturer.To monitor plasminogen uptake by cells and tissues eviously . The purn = 3 rats per group  or the saline control group (C)  all CSF samples were immediately flash-frozen in liquid nitrogen and stored at −80°C until use.The anaesthetized rats were placed in a stereotaxic frame and secured with ear bars, and a midline incision in the skin was made up to the head area to permit easy access to the cisterna magna. The needle, which was connected to a draw syringe, was inserted horizontally and centrally into the cisterna magna for CSF collection. The CSF sample was slowly drawn into the syringe, and the colour of the CSF was closely observed to avoid any possible blood contamination. Approximately 100 μl of CSF was collected from each animal. After a short centrifugation step  treated with saline (group C1) or LPS (group L1) alone or supplemented with TXA (groups C3 and L3) received a tail vein injection of Evans blue dye  4 hours before being killed. After transcardiac perfusion with heparinized saline, the brains and kidneys were harvested and plunged into cold saline until fluorescence measurements were performed. Evans blue dye permeability was monitored by ex vivo near-infrared fluorescence (NIRF) imaging using the IVIS 200 imaging system .Rats  were collected on polylysine-coated slides and incubated overnight with a goat anti-Col IV primary antibody , which was revealed with donkey anti-goat antibody F(ab′)2 fragments linked to tetramethylrhodamine isothiocyanate (1:500). The cells were then counterstained with DAPI, mounted with Fluoprep  and observed under an epifluorescence microscope. Images were digitally captured with a Leica DM6000 microscope-coupled CoolSnap camera  and visualized with METAVUE 5.0 software . The detection of exogenously administered A488-Pg in those samples was also studied.p-nitroaniline released from CBS0065 is proportional to the amount of plasmin(ogen). The ability of TXA to impair plasminogen binding to the cell membrane was investigated as described previously [Transformation of CSF plasminogen into plasmin was measured using uPA (5 IU/ml) and a plasmin-selective chromogenic substrate (0.75 mM CBS0065), as previously described . In thiseviously .Human CSF samples  and rat CSF, urine and plasma (diluted 1:50) samples (10 μl) were electrophoresed in 8% SDS-PAGE gel under nonreducing conditions. Proteins were transferred onto a polyvinylidene fluoride membrane, and plasminogen was revealed using a monoclonal antibody directed against human plasminogen  or a rabbit anti-mouse plasminogen polyclonal antibody (rat samples). The amount of plasminogen was expressed as the number of pixels directly detected using ImageQuant TL 7.0 image analysis software with the ImageQuant LAS 4000 imaging system  and expressed as nanograms per microlitre by reference to a known amount of plasminogen  electrophoresed under similar conditions. In rat samples, the amount of plasminogen was expressed as the number of pixels directly detected using the image analysis software.U test was used to compare values obtained in treated versus control rats. For human experiments, one-way analysis of variance (ANOVA) was performed to compare the patient groups. Bonferroni’s multiple-comparisons posttest was run for the pairwise comparison of groups. Statistical significance was set at P < 0.05. A specific statistical package for exact nonparametric inference ; StataCorp, College Station, TX, USA) was used.Data are representative of at least three independent experiments and are expressed as median (25th to 75th interquartile range (IQR)). The Mann–Whitney P = 0.0002; twofold increase in urine, P = 0.0071 (both by Mann–Whitney U test))   Figure A. The LPEx vivo fluorescent and NIRF images showed higher brain fluorescence in the LPS-treated rats (group L) as compared to controls (group C), indicating an increase in barrier permeability to the dye due to blood–CSF barrier impairment  or saline injection (group C2). A488-Pg appeared in the CSF and urine of all LPS-treated rats, indicating that blood plasminogen crosses the barrier during its presence in the circulation   as compared to multiple sclerosis (MS) (median = 0.3 ng/μl (IQR = 0.16 to 0.61), n = 19) and noninflammatory neurological diseases (NINDs) (median = 0.27 ng/μl (IQR = 0.18 to 0.35), n = 8) . The pairwise comparison yielded significant differences in GBS versus MS (P < 0.05) and GBS versus NINDs (P < 0.05), both analysed with Bonferroni’s posttest. GBS patients had significantly higher values of CSF total protein, albumin and IgG, as well as higher Qalb, whereas MS patients had higher index values of IgG  (see Additional file To substantiate the relevance of these s Figure . Higher in vivo data suggest that circulating plasminogen enters the CSF at the choroid plexus. This enormous epithelial/endothelial surface area is potentially available for untoward leakage of plasma proteins into CSF [Plasminogen is synthesized mainly by the liver and then is distributed to tissues via the systemic circulation . The coninto CSF ,34. Becainto CSF , we alsoBinding of plasminogen to carboxy-terminal lysine residues of membrane receptors is a well-known mechanism governing its capture and concentration onto the cell surface, where it can be either transformed into plasmin or internalized . BecauseCollectively, these findings demonstrate that, during inflammation, plasminogen is transported from circulating blood into the CSF of rats via the choroid plexus. Interestingly, the high plasminogen CSF content detected in LPS-treated versus control rats parallels the significantly higher concentration of plasminogen found in the CSF of patients with blood–CSF barrier dysfunction determined on the basis of a high Qalb. In agreement with previously published data ,32, we fAlthough there are limitations to translating results obtained in experimental animals to human diseases, our data are suggestive of a similar mechanism for plasminogen transfer from blood to CSF and may be of relevance to patients with inflammation-derived CSF barrier impairment.in vivo rat model of systemic inflammation, similar modifications of barrier function, and we also have demonstrated that plasminogen from circulating blood enters the CSF space. Altogether our data strongly suggest that the increase in CSF plasminogen concentrations detected in patients obeys a mechanism by which circulating plasminogen crosses the altered blood–CSF barrier.We have demonstrated that plasminogen is increased in the CSF of patients with a dysfunctional blood–CSF barrier. We have reproduced, in an


Acanthostega gunnari is an iconic fossil taxon exhibiting skeletal morphology reflecting the transition of vertebrates from water onto land. Computed tomography data of two Acanthostega skulls was segmented using visualization software to digitally separate bone from matrix and individual bones of the skull from each other. A revised description of cranial and lower jaw anatomy in this taxon based on CT data includes new details of sutural morphology, the previously undescribed quadrate and articular bones, and the mandibular symphysis. Sutural morphology is used to infer loading regime in the skull during feeding, and suggests Acanthostega used its anterior jaws to initially seize prey while smaller posterior teeth were used to restrain struggling prey during ingestion. Novel methods were used to repair and retrodeform the skull, resulting in a three-dimensional digital reconstruction that features a longer postorbital region and more strongly hooked anterior lower jaw than previous attempts while supporting the presence of a midline gap between the nasals and median rostrals.The early tetrapod  Ichthyostega was the first named genus from East Greenland , ..IchthyosAcanthostega skull are available , v, v23], vriptions , 11], , , . I. IAcanthAcanthostega gunnari skulls were CT-scanned for this study. All three specimens pertain to the Geological Museum of the University of Copenhagen, København, Denmark (MGUH) and are currently on long-term loan to the University Museum of Zoology Cambridge, Cambridge, United Kingdom . These specimens were collected under the 1987 auspices of the Greenland Geological Survey in Denmark; field permits were obtained by the late Dr. Svend Erik Bendix-Almgreen of the MGUH. MGUH-VP-8158 , an incomplete skull that includes incomplete, articulated lower jaws  in Tring, United Kingdom on an X-Tek H 225 ST CT-scanner. The skull table of MGUH-VP-8158, left half of MGUH-VP-8160, and skull of MGUH 29019 were scanned in 2014 in the Cambridge Biotomography Centre (Zoology Department) at the University of Cambridge on an X-Tek H225 CT scanner. Reconstruction of the lower jaws of MGUH-VP-8158 produced 1590 slices (oriented obliquely through the specimen) composed of isometric voxels with a resolution of 0.0517 mm/voxel; reconstruction of the skull table of MGUH-VP-8158 produced 1616 transverse slices composed of isometric voxels with a resolution of 0.035 mm/pixel; reconstruction of the right side of MGUH-VP-8160 produced 1489 transverse slices composed of isometric voxels with a resolution of 0.0964 mm/voxel; reconstruction of the left side of MGUH-VP-8160 produced 1893 transverse slices composed of isometric voxels with a resolution of 0.053 mm/pixel; reconstruction of the skull associated with the postcranial skeleton of MGUH 29019 produced 1915 transverse slices composed of isometric voxels with a resolution of 0.081 mm/pixel. CT scans were processed using the 3D visualization software package Avizo 7.1.1  on a 64-bit Dell Inspiron 15R laptop with 8 GB RAM. Within the segmentation editor, density thresholding was initially used to separate bone from matrix . Scans wSome limits to the data sets used in the description and 3D reconstruction should be noted. Only CT scans of MGUH-VP-8158 and the right side of MGUH-VP-8160 were fully segmented. This was due to strong distortion of the left side of MGUH-VP-8160 and dorsoventral crushing and loss of the skull table in MGUH 29019; nonetheless, both of these specimens  provided descriptive information. The left lower jaw ramus of MGUH-VP-8158 is broken at the level of the anterior limit of the mandibular adductor fossa and the right lower jaw ramus is broken at the posterior end of the anterior coronoid; this specimen was used primarily for understanding the morphology of the mandibular symphysis. The skull table of MGUH-VP-8158 is preserved to a point posterior to the parietal foramen and both lateral margins are missing. Only the parasphenoid, basisphenoid, basioccipital and left stapes are well-preserved in MGUH-VP-8160; other elements of the braincase are absent or fragmentary.Homo sapiens as a ‘template’ upon which fragments are assembled , ,  but, in ssembled ; howeverNearly all of the bones used in the 3D reconstruction are from the right side of MGUH-VP-8160, with the exception of the postparietals (see below). As MGUH-VP-8160 is mediolaterally compressed, the intramandibular angle (48°) of MGUH-VP-8158  was measured on the original specimen and used as a guide for initial reconstruction. The right lower jaw of MGUH-VP-8160 was digitally duplicated and reflected across the sagittal midline in Avizo and the lower jaws set at an intramandibular angle of 48°. Using the Transform Editor within Avizo, the teeth of the upper jaws (and their associated bones) were placed in their correct positions relative to the teeth of the lower jaws—as preserved in fossils, the premaxillary/maxillary tooth row is located external  to the marginal dentary tooth row, and the palatal tooth row fits between the dentary and adsymphysial/coronoid tooth rows. The cranium was then built upwards by fitting the remaining cranial elements together at sutural contacts. The complete left postparietal of MGUH-VP-8158—along with portions of the surrounding supratemporal, tabular and parietal—was reflected to become a right side element, scaled by a factor of 0.32 , and incorporated into the 3D reconstruction. Right side elements were duplicated and reflected to form the left half of the cranium.Acanthostega meet closely at the midline anteriorly, separating posteriorly to allow the parasphenoid to fit between them. The pterygoids must also articulate correctly with the marginal palatal bones  and the basipterygoid processes of the braincase. When the pterygoids were initially fitted into the reconstruction, they overlapped slightly in the midline, suggesting that the reconstructed skull was too narrow. The intramandibular angle was gradually increased to 53.4°, at which point the pterygoids articulated properly with each other and surrounding elements, resulting in a slightly wider and flatter skull , and middle and posterior fragments pushed laterally into the Meckelian canal and mandibular adductor fossa. These surfaces were manipulated in 3D using the Transform Editor and their edges fitted against the undeformed anterior segment. Breaks were manually repaired in the Segmentation Editor using interpolation. Additionally, the right pterygoid features a tab of bone at its lateral margin (level with the midpoint of the ectopterygoid) that has been partially broken away and deformed relative to the main body of the pterygoid. This breakage and deformation is localized and does not affect the pterygoid’s articulations with adjacent bones.Acanthostega skull” below.) Unless noted, all bones are right side elements from MGUH-VP-8160.The preserved portion of the left lower jaw ramus of MGUH-VP-8158 measures 88 mm total length; the skull table of MGUH-VP-8158 has a maximum anteroposterior length of 45 mm; the skull of MGUH-VP-8160 measures 111 mm from the anterior tip of the right median rostral to the posterior tip of right preopercular. As preserved, the orbit of MGUH-VP-8160 measures 16 mm tall (at its midpoint) and 18 mm in length.  was predicted by the shape of the nasals . T. Tpremaxby Clack , 12], , [, [Acanth present . However present  identifi feeding . Given t feeding  but of TAcanthostega supports the use of the enlarged front teeth in strongly biting and seizing prey, with feeding forces being channeled through the nasals and frontals to the skull roof . Contacts between the maxilla, cheek region and palatal bones (and the dentary and infradentaries) suggest the smaller posterior teeth were used to restrain struggling prey, possibly during ingestion. In general, we find that sutural morphology in the skull of Acanthostega is more consistent with an aquatic mode of feeding , corroborating results by Anderson et al. [In summary, sutural morphology in the upper and lower jaws of n et al.  and Neenn et al. . HoweverAcanthostega skull, including new information on sutural morphology, the anatomy of the jaw joint and implications for jaw movements, and a detailed description of the mandibular symphysis. Sutural morphology is used to infer loading regime and possible feeding behavior in this taxon, although these predictions are being subjected to further biomechanical analyses, including finite element modelling, by the authors. Lastly, CT data and visualization software, coupled with novel methodology, were used to create a 3D digital reconstruction of the skull of this iconic fossil taxon, which exhibits both similarities and differences to previous attempts.The results presented here supplement and amend previous descriptions of the S1 MatrixAll bones from the lower jaws were exported in a single piece. Reassembled bones from the right side of the skull were mirrored and transformed in a single piece.(DOCX)Click here for additional data file.S1 ModelDownload the PDF file and click once on the skull to activate. Left-click to rotate the model; right-click to zoom in or out; and hold both buttons to pan. Check or uncheck boxes in the tree on the upper left corner of the viewer to display or hide individual parts.(PDF)Click here for additional data file.


On September 12, 2014, CDC was notified by the Colorado Department of Public Health and Environment of a cluster of nine children evaluated at Children’s Hospital Colorado with acute neurologic illness characterized by extremity weakness, cranial nerve dysfunction , or both. Neurologic illness onsets occurred during August 8–September 15, 2014. The median age of the children was 8 years (range = 1–18 years). Other than neck, back, or extremity pain in some patients, all had normal sensation. All had a preceding febrile illness, most with upper respiratory symptoms, occurring 3–16 days (median = 7 days) before onset of neurologic illness. Seven of eight patients with magnetic resonance imaging of the spinal cord had nonenhancing lesions of the gray matter of the spinal cord spanning multiple levels, and seven of nine with magnetic resonance imaging of the brain had nonenhancing brainstem lesions . Two of five with magnetic resonance imaging of the lumbosacral region had gadolinium enhancement of the ventral nerve roots of the cauda equina. Eight children were up to date on polio vaccination. Eight have not yet fully recovered neurologically.μL), predominantly lymphocytic, consistent with an inflammatory or infectious process. CSF glucose was normal; CSF protein was normal or mildly elevated. Initial testing of CSF from eight patients showed no evidence of West Nile virus antibodies, although further testing is pending. CSF testing for enteroviruses, including enterovirus D68 (EV-D68), enterovirus 71, and poliovirus, by reverse transcription–polymerase chain reaction (RT-PCR) was negative in all patients. Other CSF tests for infectious causes were unrevealing.Eight patients demonstrated a mild to moderate cerebrospinal fluid (CSF) pleocytosis  by RT-PCR.This cluster of acute neurologic illnesses occurred against a backdrop of detection of EV-D68 causing severe respiratory disease in many parts of the United States, including Colorado ,2. Therehttp://www.bt.cdc.gov/han/han00370.asp ), which provides guidance for identifying and reporting cases. Clinicians should report to their local and state health departments patients aged ≤21 years with 1) acute onset of focal limb weakness occurring on or after August 1, 2014, and 2) magnetic resonance imaging showing a spinal cord lesion largely restricted to gray matter. To prevent infections in general, persons should stay home if they are ill, wash their hands often with soap and water, avoid close contact (such as touching and shaking hands) with those who are ill, and clean and disinfect frequently touched surfaces.On September 19, the Colorado Department of Public Health and Environment issued a Health Alert informing Colorado clinicians of this cluster and requesting reports of similar cases. One additional case with similar neurologic findings was reported as a result of this advisory and is currently under investigation. On September 26, CDC issued a national Health Advisory (available at


MRI is not only beneficial in the diagnosis of cerebrospinal fluid (CSF)-related diseases, but also aids in planning the management and post-surgery follow-up of the patients. With recent advances in MRI systems, there are many newly developed sequences and techniques that rapidly enable evaluation of CSF-related disorders with greater accuracy. For a better assessment of this group of disorders, radiologists should follow the developments closely and should be able to apply them when necessary. In this pictorial review, the role of MRI in the evaluation of hydrocephalus, CSF diversion techniques, and other CSF disorders is illustrated.Teaching PointsThe 3D-SPACE seems to be most efficient technique for evaluation of hydrocephalus and ventriculostomy.• In complex cases, PC-MRI, 3D-heavily T2W, and/or CE-MRC images may prevent false results of 3D-SPACE.• MRI is beneficial in the diagnosis and management of hydrocephalus and other CSF-related diseases.•  Progressive developments in magnetic resonance imaging (MRI) technologies allow us to better assess CSF circulation. Therefore, MRI aids in the diagnosis of diseases that result from alterations of the CSF circulation. Hydrocephalus, which constitutes a major CSF-related disorder, is well demonstrated using MRI. MRI also helps to discriminate the aetiology of the disease . The proThe CSF volume is approximately 150 ml in adults; 125 ml is distributed in the cranial and spinal subarachnoid spaces and 25 ml is found in the ventricles [CSF is produced in the choroid plexus, brain parenchyma, spinal cord, and ependymal lining of the ventricles. Most is secreted in the lateral ventricles and leaves the ventricles through the foramen of Monro to enter the third ventricle. From there, the CSF flows into the fourth ventricle through the aqueduct. It leaves the fourth ventricle by the foramen of Magendie and foramina of Luschka and enters the subarachnoid space. Cerebrospinal fluid is essentially absorbed into the internal jugular system via cranial arachnoid granulations. However, multiple experiments indicate that movement along nerve roots and exiting vessels also plays a role , 3. In aHydrocephalus is a complex disorder that can develop for various reasons. Dilatation of the ventricular system may lead to loss of brain cells resulting in a variety of neurological symptoms, stroke, and sometimes even death due to pressure applied on the brain parenchyma . The cauVentriculomegaly (Evans' index >0.3),Enlargement of the third ventricular recesses and lateral ventricular horns,Decreased mamillopontine distance and frontal horn angle,Thinning and elevation of the corpus callosum,Normal or narrowed cortical sulci,Periventricular white matter hyperintensities ,Aqueductal flow void phenomenon in T2W images .The most commonly used radiological criteria in the diagnosis of hydrocephalus are given below , 13 Fig;Fig. 1HeThese criteria are not specific for hydrocephalus, and their sensitivities are poor . The golPC-MRI provides quantitative and qualitative data regarding CSF circulation. On the other hand, in the presence of complex or turbulent flow, results may be false positive or negative , 15. Ano3), which is useful in obtaining high-resolution multiplanar reformatted images without exceeding specific absorption rate (SAR) limits. Other advantages are that the technique is a flexible one allowing employment of different sequence types such as T1W, T2W, fluid-attenuated inversion-recovery (FLAIR), proton-density weighted or variant flip-angle mode T2W images. Also, the 3D-SPACE technique is non-invasive and less sensitive to artefacts [In recent years, 3D sampling perfection with application-optimised contrast using the variable flip-angle evolution (3D-SPACE) technique has been developed and shown to be useful in the evaluation of patients with obstructive hydrocephalus . The tecrtefacts –19. Our Below imaging findings in different types of hydrocephalus have been summarised.Similar pathologies may lead to obstruction in different locations. The location, aetiology and severity of obstruction are crucial in order to plan the treatment procedure . It shouThe most significant finding on MRI to discriminate between acute and chronic forms of hydrocephalus is periventricular hyperintensities on T2W or FLAIR images, which is consistent with acute interstitial oedema  Unlike the other sites of obstruction, obstruction of the foramen of Monro may lead to unilateral ventriculomegaly . TumoursAqueductal stenosis (AS) can be classified as congenital or acquired , 17. TheUnfortunately, there has been no systematic research or case series to evaluat the FVO. The literature is limited to case reports . HeavilyConditions that may lead to narrowing of the foramen magnum include osteochondrodysplasias, metabolic disorders, developmental abnormalities and Chiari malformations . A narroThe characteristic example in this group is normal pressure hydrocephalus (NPH), which manifests with gait disturbance, urinary incontinence and dementia , 23, 24.Although the causes of hydrocephalus vary in a wide range, treatment procedures are basically alike . It is still debated which group of patients would benefit more from which treatment. Today, mainly two methods are used in the treatment of hydrocephalus. The first one is the ventriculoperitoneal shunt (VPS) including treatment with catheters that are placed proximal to the obstruction to take out excess CSF. Mortality, morbidity and complication rates are high for VPS and 50 % of patients face various problems within 2 years , 6, 9. TThe CSF diversion procedures explained above are carried out according to previously determined anatomic landmarks without visualising the whole brain and intracranial structures. In some of the patients with hydrocephalus, deformation and displacement or asymmetric enlargement of ventricles can be seen, increasing the failure and complication rates in these procedures. This makes preoperative evaluation critical in patients who will undergo ETV and VPS procedures . The preoperative ETV evaluation should include detailed assessment of landmarks that the endoscope will pass through such as the ventricular system, integrity of the third ventricle, patency of the Liliequist membrane, and positions of basilar artery-mammillary bodies and basal cisterns , 29. In 3) within an acquisition time of approximately 5 min [After successful ETV, expected findings on routine MRI include regression in ventricle size, interstitial oedema and demonstration of the flow void sign in the stoma , 29. Floly 5 min  Fig. 8)3) withinly 5 min , 5. On tly 5 min .Fig. 8SaAlso, MRI is a useful method for evaluating VPS. Position and integrity of the VPS catheter and other complications  can be easily evaluated by the 3D-SPACE technique with small isotropic voxels Fig. . PC-MRI Arachnoid cysts are mostly found in the temporal fossa and cerebellopontine angle, but they may also be located intra- or periventricularly . Due to CSF leakage from the nose is defined as rhinorrhoea, while leakage from the ear is called otorrhoea. CSF leakages may be traumatic, non-traumatic or spontaneous in origin, but the evaluation algorithm is the same for all three subtypes . In all Intracranial hypotension is a disease characterised by orthostatic headaches. The cardinal finding is increased intracranial blood volume . CharactAlthough ear trauma-related CSF leakages mostly manifest with otorrhoea, in some patients a fistula between the inner ear and subarachnoid space may develop without associated otorrhoea. This may lead to sensorineural hearing loss (SNHL), which may progress to being persistent unless treated accurately. This condition is a result of posttraumatic fractures that involve the ottic capsule. NCE-MRC demonstrates a decreased fluid level of the inner air. On CE-MRC. intrathecally admistered contrast material is shown to pass into and adjacent to the inner ear .If these perivascular spaces filled with CSF reach larger sizes, it may be challenging to differentiate them from other cystic lesions , 39. HeaMRI is not only beneficial in the diagnosis of CSF-related diseases, but also aids in therapy planning and post-surgery follow-up of the patients. With the advances in MRI systems, newly developed sequences and techniques allow precise evaluation of many CSF-related disorders, the most important of which is hydrocephalus. In order to be able to better assess CSF-related disorders, radiologists should follow new technologies that enable better assessment of CSF hydrodynamics and should apply them in routine use when needed. The PC-MRI and/or 3D-SPACE methods are relatively simple for evaluating true CSF flow and determining the obstruction level. Also, these techniques provide additional physiological information. The 3D-SPACE technique seems to be the most efficient and rapid for evaluating hydrocephalus, ETV and the shunt catheter. In suspicious or complex cases, PC-MRI, 3D-heavily-T2W and/or CE-MRC images may prevent false-negative or -positive results.


The detection of atherosclerotic plaques at risk for disruption will be greatly enhanced by molecular probes that target vessel wall biomarkers. Here, we test if fluorescently-labeled Activatable Cell Penetrating Peptides (ACPPs) could differentiate stable plaques from vulnerable plaques that disrupt, forming a luminal thrombus. Additionally, we test the efficacy of a combined ACPP and MRI technique for identifying plaques at high risk of rupture.in vivo MRI and co-registered in the same rabbit aorta with the in vivo uptake of ACPPs, cleaved by matrix metalloproteinases (MMPs) or thrombin. ACPP uptake, mapped ex vivo in whole aortas, was higher in disrupted compared to non-disrupted plaques. Specifically, disrupted plaques demonstrated a 4.5~5.0 fold increase in fluorescence enhancement, while non-disrupted plaques showed only a 2.2~2.5 fold signal increase. Receiver operating characteristic (ROC) analysis indicates that both ACPPs (MMP and thrombin) show high specificity (84.2% and 83.2%) and sensitivity (80.0% and 85.7%) in detecting disrupted plaques. The detection power of ACPPs was improved when combined with the MRI derived measure, outward remodeling ratio.In an atherothrombotic rabbit model, disrupted plaques were identified with Our targeted fluorescence ACPP probes distinguished disrupted plaques from stable plaques with high sensitivity and specificity. The combination of anatomic, MRI-derived predictors for disruption and ACPP uptake can further improve the power for identification of high-risk plaques and suggests future development of ACPPs with molecular MRI as a readout. Cardiovascular disease (CVD) remains the leading cause of mortality and morbidity in developed countries . AtherosRecently, strategies employing targeted molecular probes in conjunction with various imaging techniques have been able to visualize specific biological processes in atherosclerosis, including extracellular matrix changes , macrophin vivo and ex vivo [Among the targets that were deemed to be highly active and physiologically relevant, MMPs and thrombin are of particular interest. MMPs and thrombin have been linked to both atherogenesis and plaque vulnerability . MMPs ar ex vivo ,21. MMP  ex vivo . The enh ex vivo . Thrombi ex vivo . This ca ex vivo ,24,25. Tin situ spatial and focal protease activity map in diseased tissues [In this study, fluorescent-labeled Activatable Cell Penetrating Peptides (ACPPs), which were designed to target MMPs or thrombin, were tested in an animal model of atherothrombosis. ACPPs constitute a polycationic cell penetrating peptide (CPP) domain, a neutralizing polyanion and a cleavable linker to form a hairpin structure. The linker region was specifically designed so that protease dependent cleavage creates probe activation, releases CPP which allows the fluorescent-labeled CPP adherence in localized regions of elevated protease activity, thus providing a tool to generate an  tissues ,26. We p tissues . Further tissues , but thein vivo MRI of plaques and subsequent post-mortem fluorescence imaging of ACPPs to correlate disease state with localized protease activity in individual plaque. We hypothesized that these probes would show enhanced localized probe uptake in arterial plaques that are at high-risk for disruption, and the contribution of MRI-derived predictors would further complement the ACPPs in detecting disrupted plaques.The present study makes use of an established preclinical animal model (rabbit) of atherothrombosis that encompasses the above advantages for potential future translation to human disease and which simplifies the complicated plaque classification methods into a binominal model ,15. The Peptides were synthesized in house using standard protocols for Fmoc solid-phase synthesis and all peptides were amidated at their C-termini ,28. Peptex vivo. For contrast optimization purpose, probe was allowed to circulate for 6hrs. Animals were then sacrificed by pentobarbital  and aortas was excised and extended to physiological length and pinned down on a fluorescence free sample tray for optical imaging. Finally, transverse cryosections (10 μm) of aortas were collected and stained with Masson trichrome (Sigma Aldrich) to identify cellular components and thrombi. Meanwhile, at the end of the 6-hour ACPP circulation period, tissues from thoracic aorta, liver and muscle, were harvested, homogenized. The fluorescence readings of Cy5 were taken with 630nm (excitation) and 680nm (emission) for homogenized suspensions. The details and the relevant result were shown in the supporting data  rabbits (N = 25) entered the study at 3 months old. Among which, 21 rabbits went through a 12-week atherogenic protocol, including 8 week high cholesterol diet , balloon surgery , as previously described ,30. The ing data . The timPrior to actual MRI data acquisition, an ungated coronal 3D phase contrast MR angiogram was acquired as a scout image using a T1-weighted, fast-filed echo sequence. The left renal and iliac bifurcations on the scout image served as anatomic markers to plan the following MRI acquisition, and match with fluorescence imaging and histology .In vivo MRI was using a 3 Tesla (3T) Intera Philips System . Rabbits were sedated by acepromazine , ketamine  and xylazine , and then placed in a 6-element knee coil in the supine position. The heart and respiration rate were carefully monitored during the whole MRI session. The pre- and post- trigger MRIs were performed using the same acquisition protocols. The ECG gated T1 black blood (T1BB) images with fat suppression were acquired in the transverse plane starting from the left renal bifurcation to the iliac bifurcation using the following parameters: slice thickness = 3mm, gap = 1mm, FOV = 120 x 125mm2, acquired matrix = 384x362, reconstructed resolution = 234μm2, TR = 2 cardiac cycles, TE = 10ms, acceleration factor = 15, flip angle = 90°, IR delay = 350ms, number of averages = 2, sense factor = 2, and a total scan time of about 7~8min. Then gadolinium chelate  was administrated intravenously before un-gated phase contrast MR angiography (PC-MRA) acquisition. The parameters for PC-MRA were: slice thickness = 2mm, gap = 1mm, FOV = 120x125mm2, TE = 7.4 ms, TR = 17ms, flip angle = 15°, number of averages = 2, Matrix = 128x122, acquired resolution = 977x984 μm2, reconstructed resolution = 190x190 μm2. Finally, post contrast-enhanced (CE) T1BB images were acquired 10~15 min after Gd-DTPA injection with parameters identical to the previously described T1BB imaging.lesion/VAreference. To quantify gadolinium uptake, the average intensity of the vessel wall of each slice was measured from the CE T1BB, and normalized to intensity measurement of the matched non-contrast enhanced T1BB images, which generates a Gd enhancement ratio (GdR). Post-trigger T1BB images were to help to register disrupted plaques, based on the presence or absence of mural thrombus, to following ex vivo fluorescence experiment.The pre-triggering MRI analysis was performed in ImageJ (NIH). The contours for the vessel area (VA) were manually traced slice-by-slice using the PC-MRA images. After correction for vessel tapering, the image slice with the least plaque area was chosen as a reference slice and the remodeling ratio (RR) was calculated  by VAlesex vivo aortas were first imaged in closed-view using an IVIS Spectrum imaging station . 640nm excitation was used. The emission filter was tuned to 680nm and the exposure times were set between 0.25-2s. All comparisons within each figure are presented with the same exposure time. To match MRI slices, the fluorescence images of the closed-view aorta were segmented into continuous 4mm axial segments. Each segment was assigned as disrupted or non-disrupted based on visual inspection described later. The average fluorescence signal for each segment was obtained and normalized to signal from left femoral artery  to obtain the fluorescence enhancement ratio (FER). To further clarify the major source of fluorescence signal at disrupted regions, aortas were opened longitudinally and then imaged (open-view) again with both thrombi attached and removed from the underlying plaque. During each optical image session, a reflective (photographic) image was acquired using the same geometric setting for alignment purpose. Regions of interest (ROIs) of the detached thrombi and surrounding plaques were manually traced, and signals within ROIs was averaged and normalized.The t-test. A two-tailed probability value of p< 0.05 was defined as statistically significant. The Receiver Operating Characteristic (ROC) curve was to assess the power of FER, RR and GdR in distinguishing disrupted vs. non-disrupted regions. The test accuracy was measured by the area under curve (AUC). An AUC<0.50 was defined as a test failure, and the larger the AUC the stronger the parameter’s detection power against plaque disruption. The integrated power of combining the above parameters in distinguishing plaque rupture was also evaluated. The GdR, RR and FER were first normalized to the scale of 0~10. The weighted association value was obtained using the equation: Σipredictoriweighti. Where, predictori represents the normalized value of either GdR, RR or FER, and weighti represents and percentage of contribution from the corresponding predictor with a range of 0~100% and an interval of 5%. The ROC analysis was performed for each combination of different weights. The weight combination with the largest AUC is considered as the optimized combined predictor.Data are presented as mean ± SD. Comparisons between groups were performed using the unpaired student The presence or absence of attached thrombus could be identified and documented by white-light visual inspection when opening the aorta . SegmentAtherosclerotic plaques were observed in all experimental rabbits on the high cholesterol diet (Groups: A-C). Nine atherosclerotic rabbits were studied with the MMP-ACPP, and 8 with the thrombin-ACPP probe; a total of 47 segments encompassing disrupted regions and 239 segments in non-disrupted regions were analyzed quantitatively.Representative fluorescence images (closed-view) of rabbit aortas are shown in To examine whether the high fluorescence signal at the sites of plaque thrombosis originated from the plaque or the thrombus, the aortas were opened longitudinally and the thrombi removed from their attachment sites. As illustrated for the MMP-probe in in vivo MRI images and corresponding ex-vivo measured fluorescent signal were analyzed together. To explore whether or not MRI derived predicators are complementary to ACPP measurements in disrupted regions, the uptake of gadolinium chelate and outward remodeling from When comparing each single factor, the targeted ACPPs demonstrated superior detection power. The AUC was 0.79±0.05 for the MMP probe and even higher (0.90±0.05) for the thrombin probe. The optimal cutoff threshold was 3.5/4.0 fold (MMP / thrombin), with corresponding specificities of 84.2%/ 83.2% (MMP / thrombin) and sensitivities of 80.0%/ 85.7% (MMP / thrombin). The remodeling ratio showed a smaller AUC (0.69±0.05) and the GdR showed an even smaller AUC , rather than broad and complicated histological features of vulnerability.In this study, we show that two novel, targeted fluorescent ACPPs detected disrupted plaques with high sensitivity and specificity in a rabbit model of accelerated atherosclerosis. As with any animal model, the rabbit model used here cannot completely mimic atherosclerotic disease progression found in humans. However, this rabbit model develops 6 of the 8 AHA categories for human plaques, and the disrupted plaques shared several histological features known to be associated with vulnerable plaques in humans ,31. Our Currently, various imaging methods have made advances in detecting “high-risk” plaques among stable ones in human vascular disease. In addition to traditional technology, such as computed tomography (CT) or ultrasound, which focuses on occlusion of lumen, emerging methods have started to concentrate on functional aspects of atherosclerosis. For example, fractional flow reserve (FFR) can provide valuable information about the attainable blood flow despite stenosis . PositroA crucial strategy of our study was to combine ACPP measurements and MRI derived predictors for plaque disruption, the pathophysiological end point of high-risk plaques. MRI derived measures, outward remodeling and enhanced gadolinium-DTPA uptake, have been linked to plaque destabilization in previous studies ,39,40. Hin vivo applications, and therefore, a higher likelihood for translational development. Additionally, MRI would permit the detection of probe uptake in a pixel-by-pixel manner. This feature could clarify the potential ambiguity lying in the current optic imaging setting for the source of signal increase: from elevated enzyme density, which indicates plaque destabilization, or merely from thickening of vessel wall, which is observed more often in stable plaques. Furthermore, with remodeling ratio and ACPP uptake that can be measured in the same MRI session, any systematic error caused by matching different imaging modality will be eliminated. A challenge for molecularly targeted MRI probes to overcome is the limitation of low sensitivity that has been achieved in studies with other probe designs [cat/Km for ACPP cleavage by thrombin by 55 fold in a mouse model. This increased enzyme sensitivity should lower the amount of probe needed, which in turn may lower the potentially toxicity, and facilitate translation into the rabbit model for non-invasive MRI detection of destabilized plaques. Future development of gadolinium-loaded ACPPs for the MMPs and thrombin would allow detection of enzymatic activity and vascular remodeling in a single MRI examination and could significantly enhance in vivo diagnosis of vulnerable plaques at high risk for disruption.The main limitation of using the Cy5 labeled ACPPs reported here is the necessity to remove and expose the aorta for fluorescence imaging due to the relatively low penetration depth of transmitted light. In addition, the probe contrast reaches its maximum at about the 6th hour, and the animal must then be sacrificed for imaging. This makes fluorescent imaging with a 48-hour triggering period incompatible with probe injection prior to triggering rupture. For future clinical applications, this limitation could be obviated by catheter-based imaging  or possi designs ,43. The  designs . In anot designs  an improS1 Appendix(DOCX)Click here for additional data file.S2 Appendix(DOCX)Click here for additional data file.


To assess whether an objective performance criterion for in vitro fertilization (IVF) centers can be established.A retrospective analysis of 2011 National ART Surveillance System data for 451 U.S. IVF centers, 137 of which were included in the analysis since they performed >20 fresh embryo transfers per age group and >20 fresh oocyte donor transfers. The analysis of autologous cycles was restricted to women under age 40. The main outcome measure was correlation between center-specific live birth rates (LBR) in autologous and donor oocyte cycles.55.6% donor and 46.7%, 39.1% and 28.7%  autologous cycles resulted in live births per fresh embryo transfer. Donor LBR predicted autologous LBR . Clinics with high prevalence of patients with diminished ovarian reserve had lower autologous LBR per age group (P = 0.015). Every 10% increase in donor LBR increased odds of autologous LBR above the age-adjusted national average by 68% .Since center-specific donor and autologous IVF cycle outcomes correlate, and as donor cycles reflect fewer patient covariates, they represent a first comparable performance measure between centers, allowing for internal as well as external quality control. Center-specific national reports of IVF cycle outcomes, published by the Centers for Disease Control and Prevention (CDC) and the Society for Assisted Reproductive Technology (SART), allow patients limited insights into center performances. Both reports, however, emphasize in introductory materials that center-specific outcomes should not be used to directly compare centers since severity of infertility in treated patient populations can greatly vary , 2.Interest in clinical outcome measures, which allow objective clinical performance comparisons have been increasing in the general public, among medical insurance companies and from government agencies . The twoAn optimal reporting system would account for inherent differences in patient populations, patient selection biases, clinical IVF protocols and laboratory procedures as well as neonatal and perinatal outcomes. To directly compare qualities of IVF programs accurately, assessment measures would, therefore, have to account for all of these covariates, a highly complex and currently unachievable goal.Most clinical IVF programs offer in parallel autologous programs, which utilize the patients’ own oocytes, and programs, which utilize oocytes from mostly young oocyte donors, usually under age 30 years, and with normal ovarian reserve. In contrast to autologous IVF cycles, donor oocyte cycles, therefore, reflect fewer covariates, as age and ovarian reserve of oocyte donors can be assumed to be quite uniform between centers, while still maintaining the important variability of clinical cycle management and IVF laboratory performance, both very basic quality parameters for IVF centers.In a first step, attempting to establish a model to compare performance criteria between IVF centers we assessed whether in individual programs center-specific live birth rates (LBR) in donor oocyte cycles, in principle, correlate with center-specific LBR in autologous IVF cycles. Assuming such a correlation could be confirmed, deviations in individual centers could then be used for internal as well as external quality control.We reviewed center-specific outcome data from U.S. National Assisted Reproductive Technology Surveillance System (NASS) reported by 451 centers for IVF cycles initiated in 2011 (non-donor and donor oocytes), which progressed to fresh embryo transfers [Outcome data reported in the NASS system for patients utilizing non-donor oocytes, going forward here described as autologous cycles, are stratified by female ages <35, 35–37 and 38–40 years.One-hundred-and-thirty-one (131) centers were analyzed for female age <35, 124 centers for the 35–37 age group, and 119 centers for ages 38–40. Since most of the included clinics overlapped a total of 137 clinics were analyzed. LBR resulting from fresh embryo transfers from donor oocytes in those centers served as the predictor variable.Since donor cycles usually involve only carefully selected, young and healthy donors, they remove significant patient covariates from consideration, which in autologous IVF cycles can greatly differentiate treated patient populations at different centers. Yet, at the same time, oocyte donor cycles are still subject to variations in clinical management and embryology laboratory performance between centers.Donor IVF cycle outcomes, therefore, should approximate best possible LBR for all centers. Despite important potential limitations, including directed oocyte donations involving older donors and endometrial receptivity issues , both deLBR per fresh transfer was tabulated for each age group and for donor transfers in each included clinic. Comparisons of groups utilized logistic regression with a bivariate outcome variables, coded 1 for above average LBR for an age group and 0 for lower than average LBR. The actual donor LBR per center was considered the predictor variable. These results were then adjusted for the percent of patients with a diagnosis of diminished ovarian reserve (DOR) reported by each center. DOR, which diminishes LBR at all ages , was conTable Among the analyzed IVF centers, the average donor cycle LBR per fresh transfer was 55.6% (95% CI 53.7 to 57.6). The LBR per fresh transfer in autologous IVF cycles was 46.7% (95% CI 45.1 – 48.4) for women <35 years, 39.1% (37.4 – 40.9) for the 35–37 age group and 28.7% (27.1 – 30.3) for women at ages 38–40 years. Binary logistic regression analysis of center-specific donor LBR with autologous center-specific LBR as the dependent variable, adjusted for the center’s percentage of women with DOR and for age (group) demonstrated that increasing percentages of DOR patients significantly decreased odds of having above average autologous IVF LBR (P = 0.015). Every 10% increase in a center’s donor LBR increased the center’s odds of having an autologous IVF cycle LBR above the age-adjusted national average by 68% .Since donor LBR and patient age groups significantly interacted (P = 0.035), we also investigated separate models for each age group. As Table 2 = 0.18, F = 14.9, P < 0.001; 35 – 38 years, R2 = 0.13, F = 11.7, P < 0.001; 38 – 40 years, R2 = 0.05, F = 7.13, P = 0.001).Figure As stated by administrative bodies of both currently existing U.S. national reporting systems for IVF cycle outcomes, they should not be used to compare individual centers , 2 becauThe utilization of oocyte donation cycles in the U.S. has in recent years rapidly increased . Most IVThe validity of here proposed concept is further supported by the also expected observations that the prevalence of DOR in autologous cycles is statistically associated with center-specific LBR. DOR is well recognized to diminish LBR at all ages . DOR diaCenter specific LBR in donor/recipient cycles, thus, appear to represent a first tool to compare the clinical performance of IVF centers more objectively. It, however, represents only a first step, as currently nationally reported donor cycles still do include atypical cases, mostly involving directed (“open”) egg donation, where standard donor selection criteria often are waved by recipients and, therefore, older women serve as egg donors . A centeWhile IVF success is disproportionally depending on the age of the contributing female and her oocyte quality , the impHere demonstrated statistical correlation between center-specific IVF outcomes in donor and autologous cycles, therefore, can be seen as a first building stone in developing a fair, yet functionally more reliable reporting system for IVF cycle outcomes. This correlation, however, also allows for the establishment of “outliers”, which can serve centers as an internal quality of care parameter, leading them to a better understanding of where potential quality of care problems may be located at their center.th percentile, can be, likely, assured about the center’s laboratory performance but should start wondering about the center’s clinical management of autologous cycles, unless the center serves a highly unfavorable patient population.With expectations being that donor and autologous LBR correlate, centers that do not demonstrate such correlation should be able to deduct problem areas. For example, a center with donor LBR at national median but autologous LBR at only national 10th percentile in donor cycles, one has to wonder about egg donor selection and/or about, possibly, having good autologous IVF cycle outcomes only because of careful patient selection or too generous embryo numbers transferred.At the other extreme, a center at national median in autologous cycles but at national 10In an attempt to better understand the potential of here presented data for internal and external quality control, our center assessed outlier clinics, which do not demonstrate the here reported correlation between donor and autologous cycles in national reports. Our preliminary findings were recently presented  and willSince center-specific donor and autologous IVF cycle outcomes correlate, and as donor cycles reflect fewer patient covariates, they represent a first comparable performance measure between centers, allowing for internal as well as external quality control.


Decoupled oxygen-hafnium isotope systematics suggest a complex source for these magmas involving variable amounts of differentiated mantle-derived melt, Archean crust and hydrothermally altered shallow-crustal rocks. These data demonstrate that complex magma reservoirs with multiple sub-chambers are a common feature of rift- and hotspot related supervolcanoes. The short duration of reservoir assembly documents rapid crustal remelting and two to three orders of magnitude higher magma production rates beneath Yellowstone compared to continental arc volcanoes. The short pre-eruption storage times further suggest that the detection of voluminous reservoirs of eruptible magma beneath active supervolcanoes may only be possible prior to an impending eruption.Large-volume caldera-forming eruptions of silicic magmas are an important feature of continental volcanism. The timescales and mechanisms of assembly of the magma reservoirs that feed such eruptions as well as the durations and physical conditions of upper-crustal storage remain highly debated topics in volcanology. Here we explore a comprehensive data set of isotopic  and chemical proxies in precisely U-Pb dated zircon crystals from all caldera-forming eruptions of Yellowstone supervolcano. Analysed zircons record rapid assembly of multiple magma reservoirs by repeated injections of isotopically heterogeneous magma batches and short pre-eruption storage times of 10 While MFT consists of a single ignimbrite flow unit, both HRT and LCT comprise three and two eruptive members, referred to as HRT-A, B and C as well as LCT-A and B, respectively1440Ar/39Ar geochronology studies suggest that LCT-B is significantly younger than LCT-A18Three major caldera forming eruptions are distinguished at Yellowstone and are represented by the ~2.1 Ma Huckleberry Ridge Tuff (HRT) with a total erupted volume of more than 2500 kmin-situ geochemical methods coupled with high-precision geochronological techniques applied to the crystal cargo of ancient eruptions provide significant insights into the mechanisms and durations of magma accumulation prior to caldera-forming eruptions20212223192122in situ oxygen isotope and trace element analyses, hafnium isotope analyses and high-precision U-Pb geochronology, all performed on the same crystals of accessory zircon . Three crystals yielded distinguishably older dates between 2.198 ± 0.024 and 2.244 ± 0.028 Ma  flow, a normal δ18O lava , eruptedcted see  206Pb/230.028 Ma . Despite206Pb/238U dates of HRT-A zircons are statistically equivalent with a weighted mean of 2.0798 ± 0.0094 Ma (MSWD = 0.86). The excluded single crystal date of 2.212 ± 0.069 Ma corresponds to a crystal with a distinct oscillatory zoned interior domain (206Pb/238U dates and a weighted mean of 2.0794 ± 0.0047 Ma (MSWD = 1.3). Four zircons yielded statistically distinguishable dates between 2.093 ± 0.010 to 2.178 ± 0.021 Ma, reflecting either recycling of antecrystic zircons or prolonged zircon crystallization during reservoir assembly. The distribution of our zircon dates is similar to those reported for HRT-B in previous studies24et al.et al.40Ar/39Ar dates for the same unit16242740Ar/39Ar sanidine dates suggest that most analysed zircons crystallized close to eruption. These consistent data provide an excellent framework for our single crystal isotopic and chemical data and make HRT-B one of the best dated supereruptive units worldwide. The last eruption of the first Yellowstone cycle is represented by the HRT-C deposit. Eleven of twelve analyses of HRT-C zircons yielded indistinguishable 206Pb/238U dates with a weighted mean of 2.0783 ± 0.0083 Ma (MSWD = 1.3). One single analysis yielded a slightly older zircon crystallization age of 2.115 ± 0.034 Ma, equivalent to some of the older grains in HRT-B  duration27We dated zircons from all three members of the HRT. Seven of eight r domain  that hasr domain  and is iin HRT-B . The sho206Pb/238U dates with a weighted mean of 1.3004 ± 0.0073 Ma (MSWD = 1.3) that is slightly younger but overlaps with previously reported sanidine 40Ar/39Ar dates of 1.313 ± 0.11 and 1.321 ± 0.024 Ma1618O signatures  that is resolvably younger than previously reported sanidine 40Ar/30Ar dates of 0.655 ± 0.006 and 0.652 ± 0.012 Ma for the same unit1640Ar/39Ar dates of 0.615 ± 0.008 MaZircons from members A and B of LCT yielded simple distributions of Collectively, these data support previous claims of rapid magma assembly and short duration of pre-eruption shallow crustal magma storage beneath “hot and dry” Yellowstone-type supervolcanoes22213132The oxygen and hafnium isotopic compositions of Yellowstone zircons demonstrate the co-existence of multiple isotopically distinct eruption-feeding magma reservoirs at the resolution of our geochronology and their batch assembly histories in the shallow crust. Zircons from the SRB rhyolite and the three HRT members cluster in two distinct populations in hafnium-oxygen isotope space . Zircons3435371418O signature of the majority of these zircons and the lack of significant inter- and intracrystal oxygen isotopic variations suggest that assimilation of variable amounts of Archean crust occurred without recycling of low-δ18O hydrothermally altered material, and therefore likely deeper in the crust.HRT zircons within each of the two main clusters also display resolvable intercrystal hafnium isotopic heterogeneities  suggesti18O signature of the majority of MFT zircons fingerprints shallow-crustal re-melting of meteoric-hydrothermally altered intracaldera rocks2022233739MFT zircons display inter- and intracrystal isotopic heterogeneity with respect to both oxygen and hafnium isotopes . The lowZircon populations from the two LCT members are distinct with respect to their oxygen and hafnium isotopic compositions  suggestiConclusively, the paired single crystal oxygen and hafnium isotopic data highlight the importance of isotopic heterogeneities in tracing the assembly of upper-crustal supervolcanic magma reservoirs from various smaller-scale magma batches that are mixtures of multiple sources. These data also highlight the complexity of these eruption-feeding reservoirs, which comprise multiple co-existing but physically separated sub-chambers.3 to 104 years  reference zircons yielded within-session reproducibility between ±0.14 and 0.20‰ (2σ) that was propagated into the uncertainty of samples yielding an average single spot uncertainty of ±0.25‰. Analyses of secondary reference zircon Temora-2 . For samples, we preferentially targeted distinct core and rim domains identified in CL images to assess intracrystal isotopic zoning using a 15 μm probe diameter.Zircons were separated from samples of all major eruptive units of the three supereruptive cycles of the Yellowstone volcanic field using conventional techniques. 30–50 zircons per sample were selected under a binocular microscope, placed in quartz crucibles and annealed at 900 °C for 48 hours. After annealing, zircons from SRB-2, HRT-A, B, C, MFT-1 and LCT-B were mounted in epoxy resin together with natural reference materials Mud Tank and Temora-2, ground and polished to expose crystal interiors and internal textures were characterised using back-scattered electron (BSE) and cathodoluminescence (CL) imaging employing a scanning electron microscope. Oxygen isotope analyses were performed at the Canadian Centre for Isotopic Microanalysis, University of Alberta, employing a Cameca IMS1280 secondary ion mass spectrometer similar to those reported in ref. The same spots or domains were targeted for trace element analyses by laser ablation inductively coupled plasma mass spectrometry (LA-ICPMS) at the University of Lausanne employing a NewWave excimer laser ablation system attached to a Thermo ELEMENT2 ICP-MS and using NIST 612 glass as the primary reference material. Repeat analyses of Mud Tank zircon standard yielded external reproducibilities (2 R.S.D.) between 2.9% (Hf) and 18.1% (Ti). Additionally, exposed zircon-hosted melt inclusions (n = 5) were analysed for Th and U to quantify relative Th-U partitioning between zircon and melt. Analyses were performed with a 10 μm diameter laser beam and a fast scanning routine with static magnet to reduce settling times and reduce the depth of penetration of the laser.in-situ analyses, zircons were recovered from the epoxy mount, carefully cleaned in 3.5 N HNO3, transferred to 200 μl Savillex microcapsules and chemically abraded202Pb-205Pb-233U-235U tracer solutiont of 5.80 ± 0.20  and −3.33 ± 0.26 , respectively.After Zircons from LCT-A and additional crystals from other units were only analysed for U-Pb and hafnium isotopes following the protocols outlined above. Details of the analytical protocols and mass spectrometry as well as full data tables and related additional figures are given in the How to cite this article: Wotzlaw, J.-F. et al. Rapid heterogeneous assembly of multiple magma reservoirs prior to Yellowstone supereruptions. Sci. Rep.5, 14026; doi: 10.1038/srep14026 (2015).


The polymorphism of Interferon-lambda3/Interleukine28B (IFNL3/IL28B) rs 12979860 has been described as important in the development of HTLV-1-associated myelopathy/spastic paraparesis (HAM/TSP). Recently, the dinucleotide polymorphism, IFNL4 ss469415590 has been discovered and is in high linkage disequilibrium with rs12979860. In a transversal study, we aimed to examine the polymorphisms of these two nucleotides in our HTLV-1 Afro-Caribbean population.The frequencies of the CC, CT and TT genotypes of the single nucleotide rs12979860 and the frequencies of ΔG/ΔG, ΔG/TT and TT/TT genotypes of the dinucleotide ss469415590 are reported in the entire HTLV-1 group and compared between asymptomatic individuals and HAM/TSP patients.In our 94 HTLV-1 subjects, frequencies of rs1299860 were CC, 13.3%; CT, 44.7%; TT, 42% and frequencies of ss469415590 genotypes were ΔG/ΔG, 45.7%; ΔG/TT, 42.6%; TT/TT, 11.7%. We found no significant difference in allele distribution in both studied nucleotide polymorphism between 53 asymptomatic carriers  and 41 HAM/TSP patients .In our population, the polymorphisms of IFNL3/IL28B rs12979860 and IFNL4 ss469415590 are not associated with HTLV-1 neurological phenotype status. Different genotypes target should be considered.


Borrelia burgdorferi–specific antibody production (increased antibody index (AI)). However, AI can be negative in patients with early LNB and, conversely, can remain elevated for months after antibiotic treatment. Recent studies suggested that the chemokine CXCL13 in the cerebrospinal fluid (CSF) is a biomarker for active LNB. Also, CSF neopterin-level determination has been used to assess the degree of neuroinflammation in a wide variety of diseases.Laboratory diagnosis of Lyme neuroborreliosis (LNB) is partly based on the detection of intrathecal CXCL13 concentrations were analyzed in CSF samples of 366 retrospectively identified individuals. The samples represented pretreatment LNB (38 patients), non-LNB comparison patients, tick-borne encephalitis, central nervous system (CNS) varicella zoster virus infection, CNS herpes simplex virus infection, CNS HHV6 infection, CNS enterovirus infection, and untreated neurosyphilis. The panel included also samples from patients with multiple sclerosis and other neuroinflammatory conditions. Of the LNB patients, 24 posttreatment CSF samples were available for CXCL13 analysis. Neopterin concentrations were determined in a subset of these samples.The CXCL13 concentrations in CSF samples of untreated LNB patients were significantly higher  than the concentrations in the non-LNB group , viral CNS infection samples , or samples from patients with noninfectious neuroinflammatory conditions . The use of cut-off 415 pg/ml led to a sensitivity of 100% and specificity of 99.7% for the diagnosis of LNB in these samples. CSF CXCL13 median concentrations declined significantly from 16,770 pg/ml before to 109 pg/ml after the treatment.M as the cut-off led to a sensitivity of 88.6% and a specificity of 65.0% for the diagnosis of LNB. The CSF neopterin concentrations decreased statistically significantly with the treatment.CSF neopterin concentration was significantly higher among the untreated LNB patients than in the non-LNB group. The use of neopterin concentration 10.6 nThese results clearly indicate that highly elevated CSF CXCL13 levels are strongly associated with untreated LNB. CXCL13 outperformed neopterin and appears to be an excellent biomarker in differentiating LNB from viral CNS infections and from other neuroinflammatory conditions. Borrelia burgdorferi sensu lato spirochetes , and 5) intrathecal antibody production was observed as indicated by a positive (>0.3) IgM and/or IgG antibody index (AI) using the IDEIA Lyme Neuroborreliosis kit .CXCL13 levels in 390 CSF samples (including 24 posttreatment LNB samples) were measured by using a human CXCL13 kit . The frozen samples were allowed to thaw at room temperature. The analyses were performed according to the instructions of the manufacturer. Samples with concentrations over the standard curve of the assay (>500 pg/ml) were further diluted 10 to 100 times with the calibration diluent buffer of the kit, and reanalyzed. Between the first analysis and the reanalysis, the samples were stored at −20°C.Neopterin concentrations in CSF samples were measured by using Neopterin ELISA kit  according to the instructions of the manufacturer. For neopterin-concentration analysis, 262 CSF samples (including 21 posttreatment LNB samples) were available.Tick-borne encephalitis (TBE)-specific antibodies were measured by using SERION ELISA classic TBE Virus IgG/IgM kits . For viral nucleic acid testing, total nucleic acid was extracted from CSF samples by using NucliSense easyMag extractor . Enterovirus RNA was detected with RT-PCR, as described previously . DNA forTreponema pallidum hemagglutination assay (TPHA)  were used for the detection of treponemal antibodies, and VDRL  carbon antigen agglutination test (Oxoid) was used to detect nontreponemal antibodies.The single neurosyphilis case was diagnosed by using the following assays: Enzygnost Syphilis competitive enzyme immunoassay  and U test and Bonferroni corrections. Wilcoxon signed-rank test was used to compare concentrations before and after treatment. Correlations were done by using Spearman rank-order correlations. P values of <0.05 were considered significant.Statistical analyses were performed by using IBM SPSS Statistics 22. To test for the normality of the data, the Kolmogorov-Smirnov test was applied. Differences in CXCL13 and neopterin concentrations between groups were analyzed by using the Kruskal-Wallis test followed by single pairwise comparisons with the Mann–Whitney The CSF quotients of mononuclear cells, CXCL13, or neopterin before and after treatment among the sample pairs (where the posttreatment sample was collected at the end of the treatment) were calculated as follows: pretreatment CSF sample value divided by the posttreatment CSF sample value regarding CSF mononuclear cells, CXCL13, and neopterin concentrations.CSF samples from 366 retrospectively identified individuals were available for the present study Figure . Of thesThe presenting symptom in 30 of the LNB patients was radiculitis or pain radiating to the upper or lower limbs or to the trunk. Nineteen patients had facial nerve paralysis. Thirteen patients had both symptoms. In addition, some patients had intermittent or continuous headache, eye manifestations, dizziness, or paresis. The median duration of symptoms before the first CSF sample was 30 days, with the range from zero to 120 days. LNB patients were treated with either intravenous ceftriaxone, oral doxycycline, or with a combination of both of these drugs. The non-LNB group included 239 CSF samples that were sent to our laboratory for borrelia antibody analysis, and that were found to be borrelia antibody negative. Baseline characteristics of the LNB patients and the non-LNB subjects are listed in Table Samples for the comparison groups were selected based on the conception that the associated clinical manifestations are potential differential diagnostic options for LNB. The comparison samples from patients with other CNS infections included (a) ten CSF samples from patients with TBE, (b) 19 samples from patients with CNS VZV infection; (c) 14 samples from patients with CNS HSV infection; (d) three samples from patients with CNS HHV-6 infection, (e) eight samples from patients with CNS enterovirus infection; and (f) one sample from an untreated neurosyphilis patient. Also, the comparison sample panel included 34 samples from patients with confirmed (19) or suspected (8) multiple sclerosis (MS), or with other neuroinflammatory conditions (7). In all these 34 patients, CSF oligoclonal (OC) bands were detected.P < 0.001). Among the samples of the patients with viral CNS infections, the CSF CXCL13 concentrations ranged from <7.8 to 406 pg/ml, and the statistical difference between these viral CNS infection samples and the LNB samples was highly significant, with P values of 0.0013 or less in all cases. CSF CXCL13 concentrations in the 34 samples from patients with noninfectious neuroinflammatory conditions ranged from <7.8 to 280 pg/ml, which was also significantly different (P < 0.001) from the CXCL13 levels of LNB samples.The concentrations of CXCL13 in CSF of all untreated LNB patients ranged from 424 to 158,000 pg/ml, with the median of 6,480 pg/ml  curve analysis  shown in Figure Neurosyphilis caused by the spirochete P < 0.001) among the untreated LNB patients  than in the non-LNB group . Conversely, and in contrast to CSF CXCL13 levels, neopterin concentrations were widely scattered over the entire measuring range in LNB, TBE, VZV, and HSV samples. In fact, CSF neopterin levels were significantly higher among the VZV (P < 0.05) and HSV (P < 0.01) infected patients than among LNB patients. In the single neurosyphilis sample, the neopterin concentration was 92.2 nM. Among the samples of the other neuroinflammatory patients, CSF neopterin levels were uniformly low.ROC curve analysis  was performed also with the neopterin data, and the result is shown in Figure Neopterin concentrations were analyzed in the previously mentioned sample groups, although in most of the groups, the number of samples available for neopterin analysis was somewhat smaller than the number of samples in CXCL13 analysis Figure . NeopterP < 0.001). Among the five posttreatment samples that were collected only months after the treatment, CXCL13 was <7.8 pg/ml in four samples and 20.6 pg/ml in one sample.Twenty-four posttreatment CSF samples were available for CXCL13 analysis from the confirmed LNB cases. Nineteen of the samples were collected at the end of or right after the antibiotic treatment. Five samples were collected several months after the first sample. The decline in CSF CXCL13 levels along the treatment was striking; the median concentration before the treatment among the 24 samples was 16,770 pg/ml , and after the treatment, 109 pg/ml  Figure . The decP < 0.001). The median concentration before the treatment was 47.9 nM and after the treatment, 7.5 nM .Also, the CSF neopterin concentrations  decreased statistically significantly along with the treatment  in LNB patients. In non-LNB patients, the levels of CXCL13 varied from <7.8 to 153 pg/ml. The single patient with neurosyphilis had a CXCL13 level of 37,000 pg/ml. Neurosyphilis is known to elevate the level of CXCL13 in the CSF [Previous studies reported different cut-off values of CSF CXCL13 in LNB ranging from 61 pg/ml to 1,229 pg/ml ,12,13. TThe LNB patients were treated either with intravenous ceftriaxone, oral doxycycline, or with a combination of both, according to Finnish practices and international recommendations . Levels Trypanosoma brucei gambiense infection [Elevation of CSF neopterin is a marker of an acute or active CNS inflammation, but it is not specific to any particular disease . Neopternfection . In our P value 0.057. In a previous study, a significant correlation occurred between CSF mononuclear cells and CXCL13 [In statistical analyses, no significant correlations were found between different CSF parameters in the pretreatment LNB samples, or in the quotients of CSF mononuclear cells, CXCL13, or neopterin before and after the treatment. Only in the correlation of CSF neopterin concentration versus CSF cell count was a trend noted toward significance with Some limitations exist in the current study. First, because of the nature of the study, the samples were mainly from preselected hospital patients, and no CSF samples of healthy people were included. The borrelia antibody-negative non-LNB samples were collected during diagnostic workup from patients with some degree of neurologic symptoms, and thus they do not represent healthy people. Also, the untreated LNB patients were older  than non-LNB subjects . However, CSF CXCL13 concentrations among these non-LNB samples were uniformly low, highlighting the specificity of elevated CSF CXCL13 as a biomarker for LNB.Borrelia burgdorferi in the CSF and serum, and their CSF CXCL13 concentrations ranged from 498 to 8,600 pg/ml, suggesting also that these patients were most likely true LNB patients. Also, the clinical manifestations among the probable LNB patients  fit in with the diagnosis of LNB. No follow-up samples were available of the probable LNB patients, who had normal AI in the pretreatment CSF sample, and thus it remains unsolved whether the AI would have turned positive later during the infection.Second, seven patients represented probable LNB cases based on normal or not determined AI, or on lack of CSF pleocytosis. However, all these patients did have antibodies against et al. unpublished data).Third, sample storage at −20°C before testing may in principle decrease levels of CXCL13, and thus result in falsely low concentrations . Some ofFourth, no CSF samples of patients with purulent meningitis, CNS lymphoma, cryptococcosis, African trypanosomiasis, or HIV were among the comparison samples. Previously, it was reported that CSF CXCL13 may be elevated in patients with these conditions ,8,13,28.The results of the present study suggest that CSF neopterin is an insensitive and unspecific biomarker that is not suitable for diagnostics of LNB, but it may have role in distinguishing neuroinfective diseases from neuroinflammatory states. In contrast, CSF CXCL13 appears to be an excellent biomarker in differentiating LNB from viral CNS infections and from other neuroinflammatory conditions when locally determined cut-offs are used. It is also a useful tool for follow-up of LNB patients after antibiotic treatment. However, CSF CXCL13 results should be interpreted in conjunction with intrathecal borrelia antibody production, CSF pleocytosis, and ideally also borrelia nucleic acid amplification result. Limited information is available of CSF CXCL13 levels in chronic LNB , or in rTreponema pallidum hemagglutination assay; VDRL: Veneral Disease Research Laboratory; VZV: varicella zoster virus.AI: Antibody index; CNS: central nervous system; CSF: cerebrospinal fluid; CXCL13: chemokine (C-X-C motif) ligand 13; CXCR5: chemokine (C-X-C motif) receptor 5; HHV-6: human herpesvirus 6; HIV: human immunodeficiency virus; HSV: herpes simplex virus; LNB: Lyme neuroborreliosis; MS: multiple sclerosis; NS: neurosyphilis; OC: oligoclonal; ROC: receiver operating characteristic; TBE: tick-borne encephalitis; TPHA: The authors declare that they have no competing interests.JH designed the study, supervised the chemokine analyses, analyzed the data, and drafted the manuscript. EK and JO participated in the design of the study, collected the clinical data, and helped to draft the manuscript. JP helped to interpret the data and to draft the manuscript. MW was responsible for the viral analyses and drafted parts of the manuscript. JS performed the statistical analyses and drafted parts of the manuscript. All authors read and approved the final manuscript.