MSCs). Given that hypertrophic chondrocytes can differentiate into osteoblast and osteocytes and contribute to endochondral bone formation, the effect of Midazolam on chondrogenesis remains unclear. In this study, we applied a human MSC line, the KP cell, to serve as an in vitro model to study the effect of Midazolam on chondrogenesis. We first successfully established an in vitro chondrogenic model in a micromass culture or a 2D high‐density culture performed with TGF‐β‐driven chondrogenic induction medium. Treatment of the Midazolam dose‐dependently inhibited chondrogenesis, examined using Alcian blue‐stained glycosaminoglycans and the expression of chondrogenic markers, such as SOX9 and type II collagen. Inhibition of Midazolam by peripheral benzodiazepine receptor (PBR) antagonist PK11195 or small interfering RNA rescued the inhibitory effects of Midazolam on chondrogenesis. In addition, Midazolam suppressed transforming growth factor‐β‐induced Smad3 phosphorylation, and this inhibitory effect could be rescued using PBR antagonist PK11195. This study provides a possible explanation for Midazolam‐induced congenital malformations of the musculoskeletal system through PBR.Midazolam, a benzodiazepine derivative, is widely used for sedation and surgery. However, previous studies have demonstrated that Midazolam is associated with increased risks of congenital malformations, such as dwarfism, when used during early pregnancy. Recent studies have also demonstrated that Midazolam suppresses osteogenesis of mesenchymal stem cells ( Compared with other types of anaesthetics, pharmacokinetics and the drug mechanism of benzodiazepine are well understood.A, GABAB and GABAC receptors. Among them, the GABAA receptor is of particular significance for psychopharmacology because it contains a variety of binding sites at which behaviour‐modifying drugs act to produce some or all of their effects.Benzodiazepines are widely used sedative drugs for applying regional anaesthesia.In addition to the central nervous system, benzodiazepines act on peripheral tissues performed with peripheral benzodiazepine receptor (PBR) or mitochondria translocator proteins.Although numerous papers have proved the safety of benzodiazepine and its derivatives, there remain in vitro and in vivo studies indicating the toxic effects that can occur after benzodiazepine treatment.As mentioned above, benzodiazepine and its derivatives are associated with birth defects and cleft lip/palates22.12 atmosphere and medium was changed twice a week. Only early passages (passages 3‐6) of primary hMSCs were used in this experiment. To induce chondrogenic differentiation, KP cells or hMSCs were treated with chondrogenic induction medium .5 cells per pellet were placed in a 15‐ml centrifuge tube and centrifuged to produce a pellet. The medium was changed every 3 days. For high‐density cultures, cells were seeded at 3 × 104 cells/cm2 overnight and the medium was replaced with chondrogenic induction medium the following day.KP cells, an immortalized human MSC line, were established from primary human MSCs, which were transduced using a retroviral vector LXSN‐16E6E7.2.2Cells in the pellet culture or at high densities were rinsed with PBS and fixed with 4% paraformaldehyde for 10 minutes. After fixation, cells were rinsed twice with PBS and then stained with 1% Alcian blue for 30 minutes and rinsed in PBS until the blue colour was removed from the negative control cells. The amount of Alcian blue‐stained glycosaminoglycan (GAG) was quantified as per Woods et al, with modification, by dissolving the stained GAG in 6 mol/L guanidine hydrochloride overnight at 4°C. The absorbance of dissolved stained GAG was then quantified using a spectrophotometer at OD 620 nm.2.3g for 30 minutes at 4°C and supernatant was collected in an eppendorf tube and stored at −80°C. Protein concentration was assessed using a bicinchoninic acid protein assay (Bio‐Rad) as per the manufacturer's instructions. Fifteen to thirty μg of protein was resolved using SDS‐PAGE, followed by electro‐transferred onto a methanol‐soaked polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked with 5% milk or 2% bovine serum albumin (for phosphorylated protein) in Tris‐Buffered Saline‐Tween 20 (TBST) and then incubated with primary antibody overnight at 4°C. The membrane was then rinsed with TBST and the immunocomplex on membrane was detected using horse‐radish peroxidase‐conjugated secondary antibody, and the final immunocomplexes were visualized using fluorography with an enhanced chemiluminescence reagent .After treatment, cells were rinsed twice using chilled PBS and then lysed and collected using a radioimmunoprecipitation assay buffer (Thermo) plus protease and phosphatase inhibitor cocktails (Thermo). After sonication, cell lysate was centrifuged at 15 000 × 2.4Cells in micropellet cultures were fixed with 4% paraformaldehyde, washed in PBS and then sectioned and frozen (LEICA CM 1950). The 10‐μm‐thick sections were rinsed twice in PBS, followed by soaking in SuperBlock blocking buffer (Thermo) for 1 hour at room temperature, and then incubated with primary antibody overnight at 4°C. The sections were then rinsed twice with PBS and stained with secondary antibody conjugated with Alexa 594 (Thermo). Images were taken using an Olympus epifluorescence microscope. For high‐density cultures, cells were rinsed twice with PBS and then fixed with 4% paraformaldehyde, soaked in SuperBlock blocking buffer (Thermo) for 1 hour at room temperature and then incubated with primary antibodies overnight at 4°C. The sections were then rinsed twice with PBS and stained with secondary antibody conjugated Alexa 594 (Thermo). Images were taken using an Olympus epifluorescence microscope or confocal microscope (Olympus FV‐1000).2.5Small interference RNA (siRNA) (ON‐TARGETplus™ siRNA Smartpool) specifically against human PBR was purchased from GE Dharmacon RNAi Technologies, and the transfection of siRNA (5 nmol/L) was performed using a Lipofectamine 2000 transfection reagent (Thermo Fisher Sci) as per manufacturer instructions. siRNA with a scrambled RNA sequence (siN) served as a transfection control. The protein levels of PBR in MSCs after siRNA transfection were detected using Western blot analysis.2.6P < .05.All quantified results are shown in mean ± SEM of three to four independent experiments. Statistical analyses were performed using ANOVA followed by Tukey's test for significant difference. Significance was accepted when 33.14 cells/cm2) where Alcian blue and type II collagen were able to be stained in chondrogenic induction medium‐treated cells (CHON)  containing chondrogenic induction medium for 14 days; the chondrogenic differentiation was examined using Alcian blue staining to stain GAGs or by immunostaining of type II collagen. Results demonstrated that without chondrogenic induction, the morphology of the micropellets showed incomplete structures and fractures in the centre of pellets (C), whereas pellets treated with chondrogenic induction medium showed a condensed morphology (CHON)  for 7 days. The chondrogenic induction successfully increased the protein levels of SOX9 and type II collagen, whereas Midazolam significantly inhibited protein levels of SOX9 and type II collagen  or with TGF‐β‐containing chondrogenic induction medium (CHON) in the presence of different doses of Midazolam . The Midazolam only group at dose of 10 μmol/L (MDZ10) was used as a negative control. The chondrogenic differentiation was analysed using Alcian blue, immunostaining of type II collagen as previously described. TGF‐β‐containing chondrogenic induction medium significantly increased Alcian blue content in micropellet cultures, and this chondrogenic induction was dose‐dependently inhibited by the treatment of Midazolam Figure . Early s3.34 cells/cm2) with or without chondrogenic induction medium in the presence of Midazolam  at the indicated time, and chondrogenic differentiation was evaluated using Alcian blue staining and immunostaining of type II collagen. Similar to the results that were obtained from KP MSCs, chondrogenic induction medium successfully induced an increase in Alcian blue staining, whereas Midazolam inhibited Alcian blue staining dose‐dependently  with or without chondrogenic induction medium in the presence of Midazolam (10 μmol/L) and co‐treated with PK11195  for 7 days. The chondrogenic differentiation was accessed using Alcian blue staining and immunostaining of type II collagen. We found that treatment of PK11195 using a 10 μmol/L dose significantly improved the Midazolam‐inhibited effect on chondrogenic differentiation  in the presence of chondrogenic medium with or without Midazolam (10 μmol/L) for 7 days. The chondrogenic differentiation was determined using Alcian blue staining  resulted in a decrease in cell number, which represented an off‐target effect of PK11195 (data not shown). We therefore performed siRNA specific to PBR to elucidate the effect of PBR in our model. The control  or siPBR (5 nmol/L) was transfected into primary hMSCs. Protein levels of PBR after siRNA transfection were determined using Western blot analysis, and the results showed the successful knockdown by siPBR of the endogenous PBR protein levels , we observed cell loss (data not shown), which may have been damaging to our conclusion. To rule out this possible off‐target effect of PK11195, we used siRNA specific to PBRs to evaluate the function of PBR on the actions of Midazolam in chondrogenesis. Twenty‐four hours after transfection of siRNA, the protein level of PBRs was significantly decreased compared with the scrambled control. Midazolam‐induced suppression of chondrogenesis was markedly rescued with siRNA‐down‐regulated PBR in hMSCs. This result further confirmed the role of PBR in mediating the inhibitory effect of Midazolam in chondrogenesis.3H]‐labelled R05‐4864 (another PBR ligand) binding sites was highest in the mitochondrial fraction, the nuclear and cytosolic fractions also contained significant amounts of these binding sites.The major signalling pathway used to induce chondrogenesis in this study was TGF‐β signalling. TGF‐β, a pleiotropic cytokine, is involved in the regulation of cell division and suppression of immune response.Taken together, our findings suggest that Midazolam inhibits chondrogenesis via PBR to inhibit TGF‐β signalling, which in turn suppresses chondrogenesis. This partially explains why this sedative drug can result in congenital malformation of newborns if a pregnant woman is treated with benzodiazepine for anxiety and insomnia. Thus, the future application of these types of drugs must be more thoroughly considered. Further studies to elucidate how these benzodiazepine derivatives interact with their receptors and cause various outcomes require more sophisticated investigation.The authors declare that there is no conflict of interest.YCC, KCW, ECS and YKW initiated the project; YCC and KCW designed the experiments and YCC conducted experiments of the project; BMH provided reagents; YCC, KCW, ECS and YKW analysed the results; YCC, KCW, ECS and YKW wrote manuscript; BMH edited the manuscript; YKW and ECS supervised the progress. All authors read and confirmed the final draft of the manuscript. Click here for additional data file. Click here for additional data file. Click here for additional data file.


Public data from ECHA online dossiers on 9,801 substances encompassing 326,749 experimental key studies and additional information on classification and labeling were made computable. Eye irritation hazard, for which the rabbit Draize eye test still represents the reference method, was analyzed. Dossiers contained 9,782 Draize eye studies on 3,420 unique substances, indicating frequent retesting of substances. This allowed assessment of the test’s reproducibility based on all substances tested more than once. There was a 10% chance of a non-irritant evaluation after a prior severe-irritant result according to UN GHS classification criteria. The most reproducible outcomes were the results negative (94% reproducible) and severe eye irritant (73% reproducible).To evaluate whether other GHS categorizations predict eye irritation, we built a dataset of 5,629 substances . The two best decision trees with up to three other GHS classifications resulted in balanced accuracies of 68% and 73%, i.e., in the rank order of the Draize rabbit eye test itself, but both use inhalation toxicity data (“May cause respiratory irritation”), which is not typically available.Next, a dataset of 929 substances with at least one Draize study was mapped to PubChem to compute chemical similarity using 2D conformational fingerprints and Tanimoto similarity. Using a minimum similarity of 0.7 and simple classification by the closest chemical neighbor resulted in balanced accuracy from 73% over 737 substances to 100% at a threshold of 0.975 over 41 substances. This represents a strong support of read-across and (Q)SAR approaches in this area. In this article, we use this data to assess eye irritation testing.In a parallel article (in vivo)  , also knin vivo) . Howeverin vivo) .in vitro methods; above this tonnage the use of the Draize test is recommended (in vitro assay that is performed on a confluent monolayer of Statens Seruminstitut Rabbit Cornea (SIRC) cells, are currently in the process of regulatory acceptance by the OECD. Several other eye irritation methods are listed in the OECD test guideline proposals of 2015 . Finally, the EPA recently published strategies for testing antimicrobial cleaning products2.Under the European chemicals legislation REACH, substances produced or imported in volumes greater than 1 ton per annum must be assessed for eye irritation potential. Substances belonging to the 1 to 10 ton per annum tonnage band should use ommended . Recent ommended  supportsommended , both baommended . Two of ommended . Two othommended  and the ommended , a cytotThe hope to develop testing strategies to replace the Draize test by combining several animal-free methods has raised expectations. Combination methods following the top-down bottom-up approach have been proposed .The number of animals used for Draize testing is fairly small compared to the more demanding tests, e.g., for reproductive toxicity , which ith Amendment to the Cosmetic Directive (76/768/EEC), now Regulation 1223/2009, banned animal testing for new cosmetic ingredients and requires non-animal alternatives for safety assessment. These pressures motivate the creation of computational and in vitro test models for eye irritation tests and others  of the former European Chemicals Bureau (ECB) and three reference substances databases (Eye Irritation Reference Substances Data Bank (ECE-TOC), the ZEBET database and the Laboratoire National de la Santé (LNS) database), which included, after a quality check of the Draize eye test data, 1,860 studies. However, this database is not publicly available.The EU 7d others . Howeverd others . Only mod others  a largerin silico as well as integrated testing strategies were evaluated only for small datasets. In December 2014, Verma and Matthews described the evaluation of an FDA/CFSAN-developed artificial neural network for the prediction of eye irritation on 2,928 substances with specificities and sensitivities in the 80-90% range  hazards and Draize endpoints .This publication analyses results of Draize experiments and related data available in ECHA chemical dossiers4 –Type: study description –Num: disambiguates repeat studies The database for these analyses was created from ECHA dossier pages as described  with outcome Type 1. The given equation is simply the definition of conditional probability. This reproducibility refers to multiple tests in potentially different labs and should not be confused with traditional inter-/intralaboratory reproducibility.We evaluate Draize reproducibility by answering, “What is the probability a Draize test outcome agrees with another Draize test outcome for the same chemical?” This question is answered by constructing conditional probabilities for each category: in vivo. Damage is scored for cornea, iris, conjunctivae and chemosis. Each ocular endpoint has subjective scoring rules described in 5 and defined by the UN GHS. OECD guideline data is interpreted according to the following rules  and a maximum value for each endpoint. The ECHA Draize studies report Draize endpoint values, thus allowing for the sum and maximum values to be found for these endpoints.Chemosis mean: chemosis mean scoresChemosis max: max of chemosis scores for substanceIris mean: mean iris scoresIris max: max iris scoresCornea mean: mean of cornea scoresCornea max: max of cornea scoresConjunctivae mean: mean of conjunctivae scoresConjunctivae max: max of conjunctivae scoresReversibility: longest endpoint reversal periodDraize GHS Category: H318 = Type 1, H319 = Type 2A, H320 = Type 2BIn addition, we derived one “reversibility” feature matching the study and endpoint with the longest reversibility time. For example, for a chemical with a chemosis endpoint that shows a reversibility period greater than 21 days we apply the value “irreversible” to the “reversibility” feature. Finally, the classification and labeling hazard value reported in the given substance’s ECHA dossier was used to define a Draize GHS category corresponding to the category of Draize response . The features for this model are described below:Decision trees constructed for prediction of eye irritation category from Draize endpoint features  and Draize GHS categories  used Weka’s J48 decision tree algorithm . BrieflySelection of PubChem fingerprints requires the mapping of EC-Numbers to PubChem chemical identifiers. The PubChem power user gateway was used for this purpose . SimilarThe chemical similarity graph was constructed using the Fruchterman Reingold algorithm as implemented by Gephi with area = 1000, gravity = 10, speed = 1.0 . This laIn addition to allowing analysis via computational models, availability of large numbers of Draize studies allows for more generalized analyses. Many substances were tested in multiple Draize studies. Approximately 25% of the 1,841 substances for which a mode eye irritation category could be extracted are irritants. The mapping of Draize results to eye irritant categories is well defined. However, the scoring of individual endpoints is of varying degrees of subjectivity, and observations of reversibility may be more reproducible than observer assessment of damage  both in terms of inter-observer variation and animal variation. Therefore, one might expect eye irritant categories more dependent on subjective features to be less reproducible.in vitro and repeated in vivo studies. In total 10,524 studies were extracted with the following characteristics:“Eye” contained in study typeMaterials and methods data existsResults and discussions data existsKlimisch reliability score of 1 or 2 , indicatInvestigation of acute eye toxicity reveals a large number of substances with relevant in vivo OECD TG 405 (Draize test), 2,076 were read-across based on OECD TG 405 results from other substances, 292 report OECD TG 437   , 41 repoty Test)  and in 4in vivo and read-across) there are only 3,420 unique substances, indicating frequent retesting of substances for eye irritation hazard , Type 2A (“reversible irritation”), Type 2B (“reversible mild irritation”) and non-irritating . With knOur analysis indicates a greater difference in observed severity between Type 2A and Type 2B than any other consecutive categories. This figure is built from 4,134 Draize studies, where the submitter’s interpretation could be mapped to a standard category. The difference, given by OECD, between Type 1 and Type 2 categories is a question of reversibility, whereas the difference between 2A and 2B is a question of severity . The indBy observing the dependency of eye irritation categories on severity data, we can speculate on the biological features of endpoint-specific data. The predication of Type 1/Type 2 on reversibility makes it unsurprising that Type 1/Type 2 categories are not well separated by severity scores. Conjunctivae scoring best separates categories and thus delivers the greatest information content. The cornea endpoint differentiates Type 1 and Type 2A more completely than other endpoints, suggesting that corneal damage repair is less probable than other endpoints. Chemosis and iris scoring show little separation between Type 1 and Type 2A, suggesting that these forms of damage are more easily repaired. The low prevalence of iris and cornea damage relative to conjunctivae and chemosis damage in the Type 2B category indicates that these endpoints are perhaps less sensitive to irritating substances. Alternatively, different Draize endpoints may be activated by different chemical/biological mechanisms.With access to the specific results of the Draize studies, it would be possible to create models of each Draize endpoint, and perhaps thereby identify potential differential mechanisms of iris, cornea, conjunctivae or chemosis damage.In order to assess the reproducibility of Draize eye irritation scoring, conditional probabilities for each category were constructed: in vitro replacement or machine learning approach.When juxtaposed to in silico models for the Draize eye test. We decided to model eye irritation category by using the follow features:Features of the Draize test Other GHS hazardsChemical similarity via substructure analysis.Having established that Draize results are reproducible only with some caveats, we next attempted to build By building a model based on features of the Draize test, we can determine whether the existing eye irritation classifications align with the rules given by GHS eye irritation hazard criteria. Modeling of Draize test results via other GHS hazards allows for consideration of redundant testing – if other GHS hazards have high positive or negative predictive value, then there is likely some potential for test reduction. Finally, analysis of Draize results via chemical substructures allows for visualization of the distribution of Draize types over the chemical universe. We should expect that sufficiently similar substances will have similar Draize outcomes. Cases where this hypothesis is not true may present opportunities to discover novel mechanisms of eye irritation or extend our understanding of the applicability of read-across.Eye irritation categories were modeled from a number of endpoint features as described in Section 2.3. Substances for this dataset were filtered from all REACH substances by selecting only those with endpoint data for every feature and only using studies that match ECHA’s “exp key acute eye toxicity” label and OECD TG 405. The resulting dataset is composed of 391 substances . A largeThese 9+1 features (see Section 2.3) and eye irritation category given for 391 substances are reduced into a classification and regression tree through the simple Quinlan approach of attribute selection via maximum information gain . An ideaThe decision tree resulting from the Quinlan approach  is seen in The relatively strong reproduction of Large datasets in ECHA dossiers can be used to identify testing redundancies and strategies. To evaluate redundancies within GHS categorizations, i.e., here whether eye irritation can be predicted from other hazards, we built a dataset of 5,629 substances classified as “irritant” if positive for H318, H319 or H320 and “non-irritant” otherwise. The resulting dataset contains 1,931 Draize irritants and 3698 non-irritants. The dataset contains “positive”, “negative”, or unknown values for 72 GHS hazards in the REACH extraction. Hazards with less than 100 positive predictions  or less than 100 negative predictions  were filtered out. Notably, the physical hazards (H200s) and environmental hazards (H400s) are not very predictive of Draize outcome . Many of the health hazards are predictive. Of the health hazards (H300s), H302 , H315 (“causes skin irritation”), H335 (“may cause respiratory irritation”) and H317  all show high positive predictive values.6, they are found to be positive for H318, H319 or H320 – indicating a disagreement between published ECHA dossiers and the C&L inventory for at least a subset of substances. Given these inconsistencies, we may expect other misclassifications of GHS hazards in the ECHA dossiers, which may explain other cases of lack of concordance . Complete access to ECHA classification and labeling data would enable development of improved datasets for predicting eye irritation. The unintuitive nature of these hazard relationships makes human misclassification inevitable.H312 “Harmful in contact with skin” has only an 80% positive predictive value for Draize hazards over 194 substances: this means 15 substances are positive for H312 and negative for H318, H319 and H320. Closer inspection of dossiers for three of these substances  reveal ECHA dossier evaluations of “conclusive but insufficient data for classification” for “serious eye damage/eye irritation”. Additionally, H318, H319 and H320 do not occur in the “hazard statements” field in these chemical dossiers. However, when these substances are inspected using the ECHA classification and labeling inventory databaseWhile these inconsistencies make modeling more difficult, we can still evaluate models by combining GHS values to predict H318, H319 or H320. To do this, we exhaustively searched all possible combinations of 3 hazards, for a total of 59,640 combinations (72!/69!*3!) and built datasets where the selected hazards had “positive” or “negative” values for each chemical in the subset. Decision trees were then built from these subsets with high-positive or high-negative predictive value.The two presented subset trees  were buiTo evaluate the effectiveness of chemical structural similarity approaches for this hazard, we created a dataset of substances with at least one Draize study and a mapping to PubChem. The resulting dataset contains 929 substances, which can be found in PubChem. In B, is only used for prediction of another chemical, A, if it has similarity ≥ T, where T is a threshold. Next we tested the naïve approach to similarity modeling by using k-nearest neighbors with k set to 1. In this approach every chemical eye irritation category is predicted via the eye irritation category of the closest neighbor. We evaluated the models by setting different thresholds for the minimum allowed similarity. A chemical, These strong balanced accuracies resulting from the simple approach of KNN with k = 1 and Tanimoto 2D structural distance lend credence to the similarity approach for chemical classification in the domain of Draize eye test classification. This represents a strong support of read-across and (Q)SAR approaches in this area, which can reduce testing with increasing confidence as larger datasets begin to cover more of the chemical universe.In our search we found no existing satisfactory (Q)SAR models for eye irritation. However, the accuracies demonstrated here show promise for a potential similarity-based approach for eye irritation.With 9,782 Draize studies on 3,420 unique substances, we created, based on ECHA’s publicly available registrations, a larger Draize dataset than any publicly available database. The fact that the ECHA database was not optimized for such data-mining creates some uncertainties as many text fields are not standardized, making queries difficult. A number of quality controls, consistency checks, and the plausibility of the overall results give this first analysis strong confidence. However, the demonstrated value of these data for the scientific community should urge a systematic publication of the REACH data.The first assessment addresses the prevalence of this health hazard and its sub-categories: 34% of the substances were eye irritants, somewhat higher than suggested in an earlier analysis of the New Substances Database of the former ECB with 17.4% eye irritants  showing The extensive re-testing of substances documented here  allowed a thorough analysis of the reproducibility of the test. They confirm the reproducibility issues already described by in vitro assays for eye irritation, toxicokinetic information or biological profiling as attempted in ToxCast7 or the Tox218 program, all of which could likely considerably boost the predictive value of the knowledge-base. Follow-up research should focus on the integration of external databases with the ECHA data to create stronger models for eye irritation.The preliminary analysis and mining of the dataset shows that there is both considerable predictivity from chemical structure  and biological activity . Neither alone has adequate accuracy to supplant the Draize test, although given the reproducibility problems of the assay, this result might actually be contested. Here, no attempt was made to use the information from chemico-physical properties, dedicated in silico approaches.Making this dataset available will allow such analysis by the scientific community. The relatively impressive predictive value of the naïve approaches attempted here, however, strongly supports read-across  and in s


Bipolar disorder (BD) is a severe and lifelong condition. Primary endogenic polygenetic forms are common. Secondary organic forms have received increasing interest recently due to the detection of immunological encephalopathies that mimic various psychiatric syndromes, including BD. However, only limited data about routine findings of cerebrospinal fluid (CSF) analyses in BD are available. Therefore, we investigated the frequency of alterations in the CSF in patients with BD and the association with autoantibodies, cerebral magnetic resonance imaging, and electroencephalography findings. CSF samples of patients with BD collected from January 1998 until December 2015 were analyzed retrospectively. Patients with preexisting causes for alterations in the CSF  were excluded. In total, 63 patients with BD fulfilled the inclusion criteria for the study. In 1.6% of the patients with BD, an increased white blood cell count was found in the CSF. Increased albumin quotients were found in 12.9% of the patients, oligoclonal bands (OCBs) in 1.6%, and increased immunoglobulin (Ig) G indices in 3.2% (OCBs were not measured in case of increased IgG indices). No significant differences in CSF findings were found between patients with manic and depressive episodes. The main findings of this open uncontrolled study are that alterations in the CSF may be found in a small, but potentially relevant, subgroup of patients with BD. These findings are discussed in light of the new concepts of mild encephalitis and immunological encephalopathy. The detection of patients with possibly secondary organic bipolar syndromes could open up new causal treatment options with immunomodulatory medication. Bipolar disorder (BD) is a severe, lifelong condition with prevalence rates of 1–4% . The undCerebrospinal fluid (CSF) analysis is the most precise method for detecting CNS inflammation. In routine CSF analysis, the total white blood cell (WBC) count, total protein concentration, albumin quotient, intrathecal immunoglobulin synthesis (IgG/A/M), IgG index, oligoclonal bands (OCBs), and lactate concentration are measured . ReportsThere is relevant evidence that inflammatory processes might lead to secondary forms of BD. However, only limited data about CSF basic diagnostic findings in BD are available. The current retrospective study was performed within this context. The aim of this project was to investigate the frequency of alterations in the CSF in BD. It was hypothesized that alterations would be found in the WBC count and BBB function, as well as that intrathecal humoral immune response would be found in the CSF of bipolar patient subgroups as markers of immunological dysbalance. In addition, CSF findings for patients with manic and depressive episodes were compared in an exploratory manner to detect possible differences.The study was approved by the local ethics committee . All measurements were part of the routine clinical workup. All patients gave written informed consent for lumbar punctures and cerebral magnetic resonance imaging (cMRI) diagnostics.The CSF samples collected from January 1998 until December 2015 were analyzed retrospectively. The information was extracted from our CSF database, which was established in previous projects . ComorbiLaboratory investigations were performed in the CSF laboratory of the Department of Neurology of the University Medical Center Freiburg.t-test for continuous variables was performed. For group comparisons  between patients with and without alterations in the CSF, a Pearson’s two-sided chi-square test was performed. A p value of less than 0.05 was considered statistically significant for the statistical analyses.All laboratory and instrument-based diagnostic findings, as well as clinical information, were entered into a Statistical Package for the Social Sciences (SPSS) database. The main findings of alterations were presented descriptively in absolute values and percentage figures. For group comparisons (for the CSF findings) between manic and depressive patients, an independent-sample The mean age of all patients was 48.35 ± 15.22 years. The gender ratio in the study was 25 men to 38 women. At the time of the lumbar puncture, the patient group comprised 18 patients with manic episodes, 35 with depressive episodes, and 7 with mixed episodes. In three patients, lumbar puncture was performed after recovery or remission of an acute manic or depressive episode; in all other patients, lumbar puncture was performed while the patients were symptomatic.In the BD group, 1.6% showed an increased WBC count. A false-positive pleocytosis due to hemolytic CSF was excluded. In 40% of the samples, an increased total protein concentration and, in 12.9%, an increased albumin quotient was found. OCBs were detected in 1.6% and increased immunoglobulin (Ig) G indices in 3.2% (OCBs were not measured in case of increased IgG indices). The CSF findings are summarized in Table In 60% of the patients, cMRI alterations were detected. In most cases, unspecific white matter lesions (44%) were detected. EEG abnormalities were found in 23% of the patients, the majority of which were intermittent generalized slow activities  and depressive symptoms (n = 35) showed no statistically significant differences in the WBC count , total protein concentration , albumin quotient , and IgG index . In addition, patients with (n = 12) and without (n = 51) clear-cut alterations in the CSF were compared. Alterations in the CSF were detected significantly more often in male patients . The cMRI pathologies  and the rate of EEG alterations  was not statistically different between the two groups.A comparison of the CSF findings in patients with manic , at the current stage of research, it was not ethically justifiable to puncture healthy controls. Therefore, we compared our findings with well-established reference values based on extensive work by Reiber et al.N-methyl-d-aspartic acid receptor (NMDAR) encephalitis showed initially abnormal findings in 80% of the cases. WBC counts were often mildly increased, and in 60% of patients, CSF-specific OCBs were detected  can be due to stimulus pleocytosis  and are often found in immunological encephalopathies , 14. WBCOne female patient , should also lead the clinician to consider an underlying encephalopathic pathomechanism. Future studies should analyze CSF findings in a prospective, controlled setting and correct for frequent influential factors . The detection of patient subgroups with infectious or immunological encephalitis could open up new causal treatment options.LTvE and DE initiated the study and conducted the data analyses. DE wrote the paper. DE and BH performed the data collection. All of the authors were crucially involved in the theoretical discussion and performance of this study. Furthermore, all of the authors read and approved the final version of this manuscript.DE and RD: none. TH: travel grants from Bayer Vital GmbH and Novartis. EP, SM, DC, and BH: none. NV: advisory boards, lectures, research, or travel grants within the last 3 years: Janssen-Cilag, Roche, Novartis, AbbVie, GSK, Medac, and Pfizer. OS: consulting and lecture fees, grant and research support from Bayer Vital GmbH, Biogen Idec, Genzyme, Merck Serono, Novartis, Sanofi-Aventis, and Teva. LTvE: advisory boards, lectures, or travel grants within the last 3 years: Eli Lilly, Medice, Novartis, Shire, UCB, GSK, Servier, Janssen, and Cyberonics.


EDW). An MLC‐based penumbra softener was developed in the developer mode of TrueBeam linacs to reduce dose gradients across the side border of EDWs. The movement of each leaf was individually synchronized with the movement of the dynamic Y jaw to soften the penumbra in the same manner along the entire field border, in spite of the presence of the dose gradient of the EDW. Junction homogeneity upon field misalignment for side‐matched EDWs was examined with the MV imager. The fluence inhomogeneities were reduced from about 30% per mm of shift of the field borders for the conventional EDW to about 2% per mm for the softened‐penumbra plan. The junction in a four‐field monoisocentric breast plan delivered to the Rando phantom was assessed with film. The dose inhomogeneities across the junction in the superior‐inferior direction were reduced from about 20% to 25% per mm for the conventional fields to about 5% per mm. The dose near the softened junction of the breast plan with no shifts did not deviate from the conventional plan by more than about 4%. The newly‐developed softened‐penumbra junction of EDW (and/or open) fields was shown to reduce sensitivity to misalignments without increasing complexity of the planning or delivery. This methodology needs to be adopted by the manufacturers for clinical use.Junctions of fields are known to be susceptible to developing cold or hot spots in the presence of even small geometrical misalignments. Reduction of these dose inhomogeneities can be accomplished through decreasing the dose gradients in the penumbra, but currently it cannot be done for enhanced dynamic wedges ( Similarly, a misalignment‐caused dose peak in an organ at risk (OAR) should be avoided too. The sensitivity to such misalignment can be reduced by softening the dose gradient in the penumbra region. While a steep penumbra is normally desired, it cannot be easily softened on a typical linac to smooth out the junction. Clinical consequences of setup and machine uncertainties on the dose distribution in the junction region were described, e.g., by Holupka et al.Fraas et al. utilized a match‐line physical wedge to soften the gradients in the penumbra of diverging fields forming a junction,Shackford et al. softened the dose gradient of the penumbra by dynamically moving the jaws, and implemented this technique for the open fields in craniospinal treatment.Hong et al. used overlapping IMRT‐planned regions instead of a side‐by‐side matching of diverging fields.Yom et al. employed field‐in‐field homogenization together with interfraction isocenter shifts to homogenize the dose distribution at the junction of diverging fields.Application of the feathering concept to craniospinal irradiation by using three isocenters without beam edge matching was described by Cao et al.Duan et al. described a technique of smoothing the junction between an open field and IMRT fields.Zeng et al.Garcia et al. applied the concept of stepped doses in sub‐PTVs to longitudinally adjacent PTVs treated with helical TomoTherapy.Wu et al. split the desired intensity (generated through optimization) in a large IMRT plan into two (or more) slightly overlapping intensity distributions such that the intensity in the junction area would be gradually reduced to zero in each region, and used the leaf sequence generator to obtain the leaf sequences.The techniques described above add to the complexity of the plans. Moreover, most of them cannot be applied to junctions of fields employing enhanced dynamic wedges (EDW).In this paper we describe development of an MLC‐based penumbra softener for 3DCRT that can be used concurrently with delivery of a combination of EDW and open fields. This design is an extension of the idea of a physical penumbra modifier, but we employ the MLC to dynamically modify the fluence instead of using a physical modifier. Use of the MLC instead of a physical modifier eliminates the need to mount the modifier at the gantry when softer penumbra is desired. The proposed solution was designed to restrict the magnitude of the dose dip or peak at a junction under 1 mm misalignment to no more than about 5%. This design goal was loosely based on the ICRU 50 recommendation to keep the dose in the PTV within 95% and 107% of the prescribed dose,2The softened penumbra fields and the conventional fields  were delivered in the developer mode on a Varian TrueBeam linac, version 2.0 MR1, with Millennium 120 MLC (with 0.5 cm wide leaves near the center and 1 cm wide leaves elsewhere). The instructions for moving the MLC leaves and the jaws during beam delivery, as well as instructions for the MV imager, were encoded into xml scripts using in‐house written Matlab scripts, and executed in the developer mode. The conventional clinical‐like plans were planned with Varian Eclipse, and delivered in clinical mode of the TrueBeam linac.2.A.2Δ can be achieved by moving (at constant velocity) the jaw forming the field border at the junction from x0 − Δ to x0 + Δ, where x0 is the center of the junction. This strategy does not work for side matching of EDW fields. The problem is explained in Fig. x = 0. Initially, the X jaw is retracted from the field by Δ. The X jaw starts moving into the field as the dynamic Y jaw (from EDW) begins closing, and the X jaw completes the movement at x = −Δ when the dynamic Y jaw completes its movement at y = ‘E’. The corresponding fluence profile across the x direction at y = ‘E’ is shown in Fig. y positions, and an example of a highly non‐uniform summed profile is shown in Fig. y = ‘M’. A triangular peak develops in the summed fluence because after the Y jaw passes beyond y = ‘M’, no more fluence is added to this profile. To obtain a flat summed profile at y = ‘M’, one could adjust movement of the X jaw such that its movement is completed at −Δ when the Y jaw reaches y = ‘M’, instead of when the dynamic Y jaw completes its movement, see Fig. y = ‘E’ is no longer flat, because after passing y = ‘M’, the remaining fluence is delivered only outside of the penumbra‐widening zone, see Fig. In a typical junction of static fields, reduction of sensitivity to misalignment from the nominal position (where the positions of 50% fluence coincide for both fields) can be accomplished through widening the penumbra of the individual fields. This is illustrated in Fig. 2Δ‐wide penumbra centered at x = 0 for any value of y , the portion of the X collimator at y = yi needs to move from x = Δ to −Δ, but at a modified time sequence. Movement of this portion of the X collimator needs to be completed precisely at the instant ti when the dynamic Y jaw begins to over‐shadow it. Considering ti depends on position yi, to provide independence of the penumbra width on yi, velocity, vi, of movement of each portion of the X collimator must be:To avoid non‐uniformities in the summed fluence profile as in Figs. dynamic = 10 cm and Ystatic = 0 cm, to obtain 4 cm wide penumbra at the junction at x = 0 cm. The open‐beam phase begins with all leaves (from the bank at the softened penumbra) over‐traveling the junction center by the half‐width of the penumbra, Δ, equal 2 cm, see Fig. c0, until reaching Δ beyond x = 0 cm. This means:MU0, and velocity c0 of the leaf is expressed in the units of cm/MU, not cm/s to reflect independence of the delivered MUs on the repetition rate (a convention used in the developer mode). A snapshot of the leaf positions in the middle of the open‐beam phase is shown in Fig. Δ (the start position of the entire sequence), they will not complete the movement simultaneously. As explained earlier, each section of the bendable collimator, i.e., each leaf, needs to move from x = Δ at the start of the dynamic phase to x = −Δ at the instant when the dynamic Y jaw over‐shadows the leaf. To accomplish this, the velocity ci of leaf i should be:MU0 and the dynamic Y jaw overshadows the leaf i at MUi. This is illustrated in Fig. y = −9.75 cm, −9.25 cm and −8.75 cm have completed their movement (after being over‐shadowed by the Y1 jaw) and the leaves at y > −8.75 cm continue traveling to their final destination. In both phases the X2 jaw trails 0.25 cm behind the slowest‐moving leaf in order to reduce the inter‐leaf leakage.We obtained the dynamic bending effect by employing the MLC. As in the conventional EDW,While the main concept is explained above, below are additional details:Because the width of each leaf is non‐zero, the MU when the dynamic Y jaw is over‐shadowing a given leaf is not uniquely defined. We defined such MU when the dynamic Y jaw meets the center of the leaf.The control points governing movement of the dynamic Y jaw, which are defined in the segmented treatment tables (STT) for each wedge, are not appropriate for implementation of the algorithm described above. Instead, we defined the control points at the MUs, for which the dynamic Y jaw meets the centers of the leaves, as seen in the beam's eye view (BEV). These MUs were linearly interpolated from the values in the STT.By design of the EDW, the dynamic Y jaw stops movement 0.5 cm from the stationary jaw. As the dynamic Y jaw cannot reach the center of the leaf in this 0.5 cm gap, movement of that leaf was chosen to mimic movement of the neighboring leaf (on the dynamic Y jaw side). The positions of the leaves permanently shadowed by the Y jaws are not important, and we set all leaves permanently shadowed by the Y jaws to the initial position of all leaves.For nominally static (non‐wedged) fields, the penumbra softening may be achieved by moving all leaves at a constant speed as a uniform front . This is similar to the open phase of the softened‐penumbra EDW, and similarly, the X jaw trails 0.25 cm behind the leaves.Due to the effects associated with the rounded endings of the leaves, it is necessary to add an offset to the leaf positions in order to optimize fluence homogeneity across the junctions. The method of adjusting this offset is described in section 2.B.x = 0 cm) formed by two softened‐penumbra fields to the corresponding fluence in the combined junction‐free field. The time‐integrated fluence was measured with the MV imager collecting data in the “dosimetry” mode. The imager was placed at its center position. The two image halves of the junction image were collected one after another without interrupting image acquisition to avoid uncertainties associated with background subtraction required prior to summing images. The fields corresponding to the largest available opening of the Y jaws during delivery of EDW were used: Y1 = 20 cm and Y2 = 10 cm. 100 MU were delivered for each field at 6 MV, 400 MU/min. The field size in the X direction of each half image was limited to ± 10 cm from the central axis (CAX), and the collimator was set to 270°. Open beams and EDW 60°, Y1‐in, were analyzed.The value of the offset added uniformly to all leaf positions  were simulated by moving the imager longitudinally between the first and the second half‐image. The moves of the imager were verified to be accurate within no more than 0.25 mm within the applicable range of movement. The same EDW fields were used as during the adjustment of the offset of the leaves.http://rsb.info.nih.gov/ij). To improve precision, each profile point was averaged along the junction within ±1 mm from the nominal position, and ±0.4 mm across the junction.The fluence profiles were read from the DICOM images from the MV imager using ImageJ 1.43 u  breast plan delivered to a modified Rando phantom . EBT3 film  was used to measure the dose distribution in the junction region. The phantom, with right breast attached, was placed on the breast board (tilted by 13°), and was wrapped with superflab layers to facilitate placement of film in approximately coronal plane at the depth of 1.5 cm and 3.0 cm, see Fig. The film irradiated in the phantom, and the dose calibration film pieces  were scanned 24 hrs after the irradiation with an Epson Perfection V700 Photo flat‐bed scanner . The color corrections were disabled, only the red color was analyzed, and 150 dpi resolution was selected. Film orientation with respect to the scanner bed was the same for all scans. The scanner bow‐tie effect along the scanner light source was neutralized by multiplying by a dose‐independent empirically‐determined function (an inverted Gaussian), similarly to Menegotti et al.33.A.In this section we demonstrate that the fluence in the junction of softened‐penumbra EDW fields and/or open fields closely resembles the fluence of a combined (junction‐free) conventional field. Such data validates that the bendable‐collimator design allows avoiding fluence inhomogeneities along the junction [as seen in Fig. The fluence profiles along the junction of the softened‐penumbra fields after adjustment of the leaf offset are plotted in Fig. 3.B.In this section we evaluate the design for sensitivity of the summed fluence to field misalignment, and demonstrate improvement over conventional junctions.The summed‐fields fluence profiles (collected with the MV imager) across the junction of side‐matched softened‐penumbra 60° EDWs are plotted in Fig. 3.C.In this section we examine a clinically‐relevant four‐field monoisocentric breast plan. The correspondence of the softened‐penumbra plan to the conventional plan is inspected, as well as sensitivity of the dose distribution to introduced shifts (away and towards the junction) of the fields forming the junctions. Reduction in sensitivity of the dose variations to shifting the fields is demonstrated over conventional junctions.The dose distribution of the softened‐penumbra junction does not need to be identical to the distribution in a conventional‐junction plan as long as it meets the planning objectives. Nevertheless, similarity of the two distributions is desired, as it will allow replacing the conventional plans with the softened‐penumbra plans. Similarity of the two plans  is demonstrated in Figs. The improvement in the dose uniformity upon misalignment of the fields in the junction between the nodal fields and the tangents is illustrated in Fig. 4The design described above met the design goal of keeping the dose changes within ±5% upon 1 mm misalignment of the fields forming the junction. As such, the design adds another option to minimize dosimetric consequences of geometrical misalignments of fields forming junctions. It should be noted that in general, this design goal cannot be achieved with conventional junctions, as demonstrated in section While the dose distribution across the junction of the softened‐penumbra junction is close to the distribution from a conventional plan, it is not identical. This should not be a problem clinically, as it is the plan with the softened penumbra that is to be evaluated for meeting the plan objectives, including dose heterogeneity inside the PTV within −5% and 7% of the prescribed dose, and absence of hot spots. In some cases, the softened‐penumbra plan may be actually preferred over the conventional plan, even in the absence of geometrical misalignments. This may be the case of four‐field monoisocentric breast plans, where the softened‐penumbra junction feathers out dose gradients in the interpectoral axillary nodes region located in the vicinity of the junction.Clinical implementation of the softened‐penumbra requires adaptation by the manufacturers of linacs and treatment planning software. In the clinical mode of Varian's TrueBeam v2.0 MR1, concurrent movement of the jaws and the MLC is not allowed. The dose distribution in the presence of simultaneous movement of the dynamic jaw of the softened‐penumbra EDW and the leaves of the MLC can be computed similarly as in the step‐and‐shoot method with the control points matching those in the softened‐penumbra design. Refinement is possible by introducing interpolated control points .This design relies on orthogonality of the junction plane to the skin at the beam entrance, and this limitation is shared with many other designs employing penumbra softening. This is because adding the doses from the ramped penumbras of the fields on the opposite sides of the junction yields a flat dose profile only under such orthogonality. This orthogonality requirement is approximately satisfied for four‐field monoisocentric breast plans. The anterior, the posterior and the medial tangent fields typically satisfy the orthogonality requirement, and the lateral tangent field approximately. The latter is true because the ratio of the tissue‐phantom ratios (TPRs) for the depths at the superior‐inferior coordinate varied by ±2 cm is typically no more than a few %. In any case, inspection of the computed dose distribution would allow confirming the absence of artifacts at the junction potentially caused by the design.Blocking OARs may be partially affected by the design because the leaves of the MLC need to move across the junction, and because the leaves used to soften the penumbra cannot be simultaneously used to block an OAR. In many cases, the leaves at the edge of the field may be used to block an OAR instead of softening the penumbra, without any noticeable influence on the junction. This is the case of blocking the spinal canal in the nodal fields of four‐field monoisocentric breast plans, where the spinal canal is entirely outside of the BEV of the tangent fields. There are no restrictions on blocking OARs using the other bank of the MLC , as those leaves are not employed to soften the penumbra. Regardless, the computed dose distribution should be examined to confirm the plan meets the planning objectives.The choice of ±2 cm as the width of the softening zone is not unique. Increasing the softening zone allows reducing sensitivity to misalignment of the fields at the junction even further, even though the value of ±2 cm allowed meeting the design goal. On the other hand, it is not recommended to extend the softening zone much beyond ±2 cm. This is in part because widening the penumbra results in enlarging the underdosed zones in the high‐dose portion of the penumbra and the overdosed zones in the low‐dose portion in the regions where the fields across the junction do not overlap. This phenomenon is not unique to this design, and it may also be present in other methods that soften a junction. Extending the width of the softening zone also reduces the maximum usable field length, which is reduced by the half‐width of the zone (2 cm here) from the machine limit . Longer fields are possible (up to 38 cm) for non‐half‐blocked fields, but couch rotation would be needed to maintain orthogonality of the junction plane to the skin, as well as subfields. It should be noted the usable field length is not restricted by the range of movement of the MLC carriage (15 cm on Varian linacs), because the leaves move only 4 cm during the sequence.To eliminate the residual inter‐leaf leakage (not blocked by the trailing X jaw) in the open‐beam phase of the softened‐penumbra EDW or softened‐penumbra nominally static fields, we considered using the X jaw instead of the MLC. Unfortunately, combining such fields into junctions introduced additional artifacts due to differences in the shape of the dose penumbra of a square jaw and round‐ended leaves of the MLC.While there is a current trend to choose IMRT or VMAT over 3DCRT, there are situations when inverse planning techniques are not practical, and junctions of conventional EDWs and open fields are used. In particular, IMRT will suffer from uncertainties in the regions where the PTV is narrow in the BEV, and the instantaneous leaf gaps are small, in which case accuracy of modeling the rounded‐leaf ends in Eclipse is reduced. Also, due to the presence of the isocenter walk upon gantry or collimator rotation, combining IMRT fields across a junction may be susceptible to dose spikes/dips at the junction when different gantry and/or collimator angles are used for the fields across the junction. Use of VMAT for some sites is arguably not ideal, e.g., for breast. This is because of unnecessary irradiation of the lung from the beam exit when the beam incidence is approximately normal to the skin.It may be argued that fractionation averages junction uncertainties, which eliminates a need for improving junction quality. This is not always true, because a systematic misalignment will not average out. In addition, there is a growing tendency to reduce the number of fractions, and consequently diminish the averaging effects from the process.Compensation for organ movement can lead to additional uncertainties at the junctions. This is the case for the junction in four‐field monoisocentric breast plans delivered during deep inhalation breath hold (DIBH). The reason is the nodal fields are delivered on a different breath hold than the tangents, and for practical reasons, the chest position  is only held within a range of positions, not at the same position on every breath. The softened‐penumbra design should reduce uncertainties related to organ‐movement.Softened penumbra field borders might also be considered when disease progresses, and a larger PTV might be needed in the future. This would happen when treating a breast as tangents, with a possibility of treating the axillary nodes in the future: the superior borders of the tangents would be softened out initially, and the nodal fields with softened inferior borders would be added in the future when required. Half‐body irradiation could also benefit from this approach through reduction of junction artifacts upon matching the opposite body half in the future. Ideally, the fields would be delivered under extended source‐to‐skin distance (SSD) with rotated couch to keep the (future) junction in the axial plane, and subfields would be employed to even out dose gradients caused by variations of the SSD. While this approach does not require use of wedges, the penumbra softener developed in this project allows delivery of open fields too.The softened‐penumbra technique may be also useful in matching long fields employing hinged wedge pairs. A hinged wedge pair offers better control of the dose homogeneity at depth over a single beam or a parallel‐opposed pair, and clinical applications could include leg sarcoma or spinal canal. The softened‐penumbra fields for treatment of the spinal canal could be also matched to the brain fields  for complete cranio‐spinal treatment with the junction(s) practically insensitive to misalignments. This would be similar to the solution of Duan et al.,Softened‐penumbra fields might also improve junction quality for junctions that are not geometrically matched, e.g., when large fields are delivered without couch rotation, and the fields are only matched at a single depth, e.g., at the skin.5A prototype penumbra softener was developed to reduce misalignment‐caused dose heterogeneities at junctions of a side border of EDWs and/or open fields in 3DCRT. This penumbra softener employs the MLC, such that the movement of the leaves is synchronized with movement of the dynamic Y jaw of the EDW. Performance was evaluated in the four field monoisocentric breast plan delivered to a Rando phantom, and approximately four‐fold reduction of the dose dips or spikes in the junction was observed when compared to the plan with the conventional junction. The time required for planning and complexity of the plan with the softened penumbra junction should be similar as for the conventional‐junction plan. The system requires adoption by the manufacturers for clinical applications.A provisional patent for the design was filed with USPTO.


African Americans have been reported to have a higher prevalence of Alzheimer’s disease (AD) than Caucasians, but etiology-specific AD biomarkers have not been systematically analyzed in older African Americans. Coexisting cerebrovascular disease may also contribute to this increased prevalence. We hypothesized that cerebrospinal fluid (CSF) biomarkers of amyloid, neurodegeneration, and endothelial dysfunction would differ between older African Americans and Caucasians with normal cognition and cognitive impairment associated with AD.181, respectively), endothelial dysfunction , α-synuclein, and neurodegeneration (neurofilament light chain [NfL]), as well as MRI markers, for hippocampal atrophy and cerebrovascular disease (white matter hyperintensity [WMH] volume).We prospectively recruited 135 older Americans to undergo detailed clinical, neuropsychological, genetic, magnetic resonance imaging (MRI), and CSF analysis from 2013 to 2015 at Emory University . We compared levels of CSF markers for β-amyloid , total and phosphorylated tau , t-tau , and Aβ40  despite similar levels of Aβ42, NfL, WMH volume, and hippocampal volume. Cognitively impaired African Americans also had lower CSF t-tau/Aβ42  and p-tau181/Aβ42 . These could not be explained by measured biomarkers of non-AD processes, but African Americans may be more susceptible than Caucasians to the cognitive effects of WMH.Sixty-five older African Americans  and 70 older Caucasians  were included. After adjusting for demographic variables, AD risk alleles, and cognitive function, older African Americans had lower CSF levels of p-tauDespite comparable levels of CSF Aβ42 and Aβ42/Aβ40, cognitive impairment in African Americans is associated with smaller changes in CSF tau markers but greater impact from similar WMH burden than Caucasians. Race-associated differences in CSF tau markers and ratios may lead to underdiagnosis of AD in African Americans.NCT02089555. Retrospectively registered on 14 March 2014.ClinicalTrials.gov,  ABCA7 ), neurodegenerative (neurofilament light chain [NfL]), and candidate endothelial markers  ) , 17 to tThis study was approved by the Emory Institutional Review Board. Informed consent was obtained from all subjects or their authorized representatives.APOE) and ABCA7 genotypes. All biochemical and MRI analyses were performed by experienced operators blinded to the subject’s race and cognitive functioning.We enrolled 65 older African Americans and 70 older Caucasians into a cross-sectional study at Emory University from 1 July 2013 to 30 June 2015. Based on our preliminary data, this sample size was sufficient to detect a difference in CSF t-tau levels. Recruitment for this study was previously described , p-tau181 levels , and Aβ40 . Race also affected the ratio biomarker of t-tau/Aβ42  according to cognitive functioning, but it had minimal effect on Aβ42/Aβ40  = 10.67; p = 0.001] and p-tau181/Aβ42  ratios than cognitively impaired Caucasians.As a group (regardless of diagnosis), African Americans had lower CSF levels of p-tau01) Fig. , t-tau (01) Fig. , and Aβ417) Fig.  than Cau17) Fig. . Univariβ42 Fig.  and p-taβ40 Fig. . For bot181 levels in African Americans may be explained by misdiagnosis, less neurodegeneration, or greater contribution from non-AD copathology toward cognitive impairment. Because CSF Aβ42 levels were indistinguishable between African Americans and Caucasians, cognitively impaired subjects in the two racial groups were equally likely to have β-amyloidopathy, which makes misdiagnosis less probable. To determine if there was a difference in neurodegeneration between the two races, we examined the relationship between race, CSF NfL levels, and hippocampal atrophy because the latter two are influenced by AD as well as non-AD disorders. ANCOVA showed that cognitively normal African Americans had lower log(NfL) than cognitively normal Caucasians , but log(NfL) did not differ between the two races for those with cognitive impairment. Race also had no effect on hippocampal volumes  to suggest less neurodegeneration in cognitively impaired African Americans. Similarly, among those with CSF t-tau/Aβ42 not consistent with AD, race had no effect on CSF α-synuclein levels  or p/t-tau ratio .Lower t-tau and p-tau181 , or Aβ40 levels . Therefore, vascular risk factors and total WMH volume do not sufficiently account for the CSF AD biomarker differences between the two races. At the same time, peripheral vascular risk factors may not adequately reflect cerebrovascular disease burden. We thus tested whether CSF levels of two candidate endothelial markers (sVCAM-1 and sICAM-1) could be used as surrogate markers of endothelial dysfunction. As a proof of principle, we first analyzed the relationship between these two markers and race, peripheral vascular risks, and WMH among cognitively normal subjects enriched for those without AD pathology . At the univariate level, CSF sVCAM-1 levels, but not sICAM-1 levels, correlated positively with older age , Caucasian race , higher WMH volume , greater number of vascular risk factors , history of congestive heart failure , and history of atrial fibrillation . ANCOVA showed that race modified the relationship between sVCAM-1 levels and WMH volume  or vascular (WMH) pathology to influence cognition (see the Methods section above). At the univariate level, cognition was influenced by APOE and ABCA7 genotypes as well as by log10(WMH) and CSF levels of Aβ42 and t-tau. Introducing the interaction term of race × log10 (WMH) showed that the interaction term had a stronger effect on cognitive Z-scores than log10 (WMH) alone (Table 10 (WMH). This was not the case for tau or p-tau181.To reconcile our finding that, after controlling for race-adjusted cognitive performance, African Americans had lower CSF tau marker levels than Caucasians despite similar levels of WMH, NfL, and hippocampal volumes, we tested if race modified the relationship between cognition and the two intermediate etiologic biomarkers . To test this hypothesis, we determined, using multivariate linear regression analysis, whether there was an interaction between race and each of these markers of AD , race was associated with lower CSF p-tau181, and Aβ40—underscores the biological and clinical importance of biomarker studies in minority populations. We believe that cognitive impairment in African Americans was indeed associated with β-amyloidopathy, because CSF Aβ42 levels in subjects with MCI or dementia did not differ between the two races. These findings imply that using existing cutoff values for CSF biomarker levels or ratios involving tau derived from largely Caucasian populations may lack sensitivity in identifying AD in minority populations, and it is not known if cerebral tau imaging can overcome this. We extended these observations by exploring multiple CSF and MRI markers involved in neurodegeneration, and we found race-independent  as well as race-dependent (sVCAM-1) markers. However, a key observation was the absence of a difference in CSF Aβ42 levels and WMH volumes between the two races. According to the model of sequential biomarker changes in AD [181 levels in cognitively impaired African Americans might result in lower CSF NfL levels and greater hippocampal volumes than similarly impaired Caucasians. This was not the case, which led us to search for an alternative explanation for the relatively greater neurodegenerative changes  in African Americans associated with more modest tau changes. Exactly how WMH may result in greater neurodegeneration and cognitive deficits in African Americans is unknown. WMH is thought to represent terminal organ damage from chronic ischemia, and it is possible that African Americans have a greater degree of subclinical endothelial dysfunction than Caucasians. It is also possible that differences in the brain structural (as measured by white matter tract integrity) and functional (as measured by resting state connectivity) networks predispose African Americans to the effects of ischemia. Finally, a synergistic effect between WMH and brain amyloid burden has been reported for cognition [Assessing cognitive decline in minority populations has been challenging because of discrepancies between various normative datasets , 35, 36.es in AD , more moognition , and braognition .MAPT or other genes (besides APOE or ABCA7) may also influence t-tau levels or the relative levels of different tau isoforms. The H2 haploptype of MAPT is rare among African, Asian, and Native American populations [MAPT variant to associate with CSF t-tau or p-tau181 levels among those without evidence of brain amyloid deposition [Our model was suggestive of a process involving WMH to account for lower tau marker levels in African Americans, but other explanations need to be considered. There still exists the possibility of greater non-AD pathology in African Americans, because CSF α-synuclein and p/t-tau are imperfect markers of Lewy body and TDP-43 pathology . Althougulations , but a pposition . Other lIn sum, we report that CSF tau biomarker levels are strongly influenced by race in a cohort of older African Americans and Caucasians in Atlanta. This has important implications for the use of diagnostic biomarkers derived from CSF and potentially other modalities  in unique populations underrepresented in existing large biomarker studies. Our data also raise the possibility that African American race may modify the relationship between an MRI marker of cerebrovascular disease and cognition, likely through an APOE-independent mechanism. There is an urgent need to test the generalizability of these findings in more cohorts of African Americans.


Endocannabinoids (ECs) are involved in immunomodulation, neuroprotection and control of inflammation in the central nervous system (CNS). Activation of cannabinoid type 2 receptors (CB2) is known to diminish the release of pro-inflammatory factors and enhance the secretion of anti-inflammatory cytokines. Furthermore, the endocannabinoid 2-arachidonoyl glycerol (2-AG) has been proved to induce the migration of eosinophils in a CB2 receptor-dependent manner in peripheral blood and activate neutrophils independent of CB activation in humans. The aim of the current study was to investigate the influence of the endocannabinoid system in two different CNS inflammatory diseases of the dog, i.e. Steroid-Responsive Meningitis-Arteritis (SRMA) and Intraspinal Spirocercosis (IS). The two main endocannabinoids, anandamide (AEA) and 2-AG, were quantified by mass spectrometry in CSF and serum samples of dogs affected with Steroid- Responsive Meningitis-Arteritis in the acute phase (SRMA A), SRMA under treatment with prednisolone (SRMA Tr), intraspinal Spirocercosis and healthy dogs. Moreover, expression of the CB2 receptor was evaluated in inflammatory lesions of SRMA and IS and compared to healthy controls using immunohistochemistry (IHC). Dogs with SRMA A showed significantly higher concentrations of total AG and AEA in serum in comparison to healthy controls and in CSF compared to SRMA Tr (p<0.05). Furthermore, dogs with IS displayed the highest ECs concentrations in CSF, being significantly higher than in CSF samples of dogs with SRMA A (p<0.05). CSF samples that demonstrated an eosinophilic pleocytosis had the highest levels of ECs, exceeding those with neutrophilic pleocytosis, suggesting that ECs have a major effect on migration of eosinophils in the CSF. Furthermore, CB2 receptor expression was found in glial cells in the spinal cord of healthy dogs, whereas in dogs with SRMA and IS, CB2 was strongly expressed not only in glial cells but also on the cellular surface of infiltrating leukocytes  at lesion sites. The present study revealed an upregulated endocannabinoid system in dogs with inflammatory CNS diseases, highlighting the endocannabinoid system as a potential target for treatment of inflammatory CNS diseases. Cannabis sativa and Cannabis indica commonly known as Marijuana to treat a variety of disorders both in humans and animals. One of the main reasons for such an effort is that numerous cannabinoids have potential medicinal effects lacking the psychoactive effects of some phytocannabinoids like ∆9–tetrahydrocannabinol (THC) [In the last decades an increasing interest emerged in the use of derivatives of the plant ol (THC) , 2. Thesol (THC)  and togeol (THC) , 5.Endocannabinoids (ECs) are endogenous lipid transmitters that mimic the action of THC by binding and activating the cannabinoid receptors . The mosCB1 receptors are expressed mostly on neurons  and mediIncreasing evidence supports the immunomodulatory roles of 2-AG and AEA . Moreovei.e. SRMA and IS. SRMA is a systemic immune disorder characterized by inflammatory lesions of vessels, particularly in the cervical leptomeninges [Spirocerca Lupi is a nematode parasite of dogs and other carnivores which affects mainly the esophagus and aorta [The aim of the current study was to investigate in a comparative way the influence of the endocannabinoid system in two different inflammatory CNS diseases of the dog, meninges . Spirocend aorta  and abernd aorta , 30. TheA total of 41 cerebrospinal fluid (CSF) samples and 36 serum samples were retrospectively analyzed. From them, 27 paired serum and CSF samples were collected between July 2008 and December 2012 from client-owned dogs diagnosed with SRMA at the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Germany. Additionally, 3 serum and 7 CSF samples from seven dogs affected with IS presented to the Koret School of Veterinary Medicine Teaching hospital, The Hebrew University of Jerusalem, Israel. Finally, 6 paired CSF and serum samples from healthy Beagles from the Department of Small Animal Medicine and Surgery, University of Veterinary Medicine Hannover, Germany, were used as controls. This study was conducted in accordance with the ethical guidelines of the University of Veterinary Medicine Hannover and was approved by the authorities of Lower Saxony . CSF samples were collected from the cerebellomedullary cistern or lumbar subarachnoidal space of the dogs under general anaesthesia, and serum samples were obtained by puncture of the cephalic or saphenous peripheral vein. Routine CSF analysis was performed immediately after collection and the remaining CSF and serum samples were stored at -20°C until further analysis.SRMA in the acute stage (SRMA A) was defined by occurrence of cervical rigidity and pain, fever and polymorphonuclear pleocytosis in the CSF . Other pSpirocerca Lupi and the combination of the following characteristic clinical and pathological findings: acute onset of non-symmetrical hind limb paralysis, radiographic or endoscopic evidences of esophageal granuloma and eosinophilic pleocytosis. Favorable response to treatment supported the diagnosis in dogs that survived and detection of the nematode within the spinal cord parenchyma confirmed the diagnosis in dogs that were euthanized due to lack of improvement.Inclusion criteria for IS required dogs to live in areas endemic for Serum and CSF samples were used for determination of both endocannabinoids (AEA and 2-AG) by stable-isotope dilution liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) , 35.A total of 8 dogs with SRMA, 2 dogs with confirmed IS, and 5 healthy controls were included in the immunohistochemical evaluation. The healthy control group consisted of 5 Beagles without clinical or pathological evidence of neurologic or infectious diseases. Tissue samples were obtained following routine necropsy and were included in previous studies , 37 condSpirocerca Lupi were sent from Korea School of Veterinary Medicine, The Hebrew University of Jerusalem, Israel.Spinal cord tissue samples from six of the 8 dogs with SRMA originated from the Department of Clinical Research and Veterinary Public Health, Vetsuisse University of Bern Switzerland, and two were obtained from the Department of Pathology, University of Veterinary Medicine Hannover, Germany. Tissue samples from dogs with confirmed intraspinal migration of Sections of the cervical, thoracic, and lumbar spinal cord were collected during necropsy and fixed in 10% formalin for at least 24 hours. After fixation, tissue samples were embedded in paraffin, cut at serial sections (3 μm), mounted onto coated slides , and stained with hematoxylin and eosin (HE). Subsequently, a complete histological examination in order to detect histopathological lesions and immunohistochemical staining to evaluate CB2 expression were performed. Given the fact that CB2 receptors are strongly expressed in immune tissues and leukocyte subpopulations , spleen Details of breed, sex, age, and morphological changes of the lesions and diagnosis from all dogs are summarized in 4-AEA  and d5-2-AG  were added to each sample, to a final concentration of 1.1 nM and 1.0 nM, respectively. This step was performed on ice. Subsequently, 500 μl Toluene  were added to each sample and homogenization was performed by shaking twice during 20 seconds at 5000 rpm at 4°C in a PreCellys tissue homogenizer. After centrifugation  the organic phase was separated and evaporated to dryness by a gentle stream of nitrogen for approximately 50 min. Finally, 50 μl eluent (25% water and 75% methanol) were added to the residues for liquid chromatographic analysis and 25 μl of the solution was injected into the Waters ACQUITY/XEVO TQ-MS LC-MS/MS system . Chromatographic separation took place on a Waters ACQUITY BEH C18 reversed phase column  [The quantification of AEA and 2-AG in CSF and serum samples was performed by stable-isotope dilution liquid chromatography combined with tandem mass spectrometry (LC-MS/MS) as previously described , 35. Brile size) , quantifle size) . The avale size) .2O2 for 30 minutes at room temperature. Slides were pre-treated with sodium-citrate buffer (pH 6.0–6. 5) in the microwave at 800w for 20 minutes for antigenic retrieval. In order to prevent non-specific binding of the antibodies, all slides were incubated with normal goat serum  for 20 min at room temperature. Following blocking, slides of interest and positive controls were incubated in a moist chamber with a polyclonal anti-CNR2/CB2 antibody  overnight at 4°C. The primary antibody used cross-reacts with human, monkey and canine CB2, as the canine CB2 sequence shares between 76 and 82% homology with rat, mouse and human CB2 [2O2 for 5 minutes at room temperature followed by a slight counterstaining with Mayer's hemalaun, dehydration in an ascending series of alcohol, cleaning in acetic acid-n-butylester , and mounting using Roti®-Histokit . Sections of tissue samples were independently examined via light microscopy  and representative images were acquired by use of photodocumentation software .Immunohistochemistry (IHC) was performed using the avidin-biotin-peroxidase complex (ABC) method as previously described , 40. Briuman CB2 . As negaStatistical analysis to quantify differences between the levels of AEA and total AG among the groups was performed using the SAS® software Version SAS 9.3 . Normal distribution of the data was found after logarithmic transformation. For descriptive statistics, measures of location and statistical dispersion were depicted as median and range due lognormal distribution of data. Group differences of endocannabinoid concentrations were tested using One-way-analysis of variance for independent samples followed by the Least Significant Difference (LSD) test, a test controlling Type I comparison wise error rate. Differences between the groups were considered significant when the corresponding p-value was lesser than 0.05. Graphics from the statistical data obtained were performed using GraphPad® Software .AEA and total AG were analyzed in 36 serum and 41 CSF samples. The distribution of the samples analyzed for AEA and total AG are summarized in The group of dogs with SRMA included 6 females (1 neutered) and 15 males (1 neutered), ranged between 6 months and 27 months of age (median 17 months). Furthermore, this group included 12 different breeds mostly Boxers (31.81%) and Bernese Mountain dogs (18.18%). In some dogs with SRMA samples were taken twice, in the acute phase (SRMA A) and during treatment (SRMA Tr). All CSF samples in SRMA A and SRMA Tr and groups were obtained from the cerebellomedullary cistern. Dogs in the SRMA A group displayed a neutrophilic pleocytosis .Seven dogs with IS were included in the study. These dogs presented with acute onset of either monoparesis (n = 2), paraparesis (n = 4) or paraplegia (n = 1) of the pelvic limbs, hyperesthesia in the lumbar (n = 2), lumbosacral (n = 1), and both cervical and lumbosacral (n = 1) regions, proprioceptive deficits of the pelvic limbs (n = 2), fecal and urinary incontinence (n = 2), reduced withdrawal reflex in the pelvic limbs (n = 1) and/or ataxia of all four limbs with hypometric gait of the thoracic limbs (n = 1). Complete blood count, chemistry profile and spinal radiographs were all normal except for elevated creatinine in one dog and mild leukocytosis in two dogs.Further evaluations included serology for Toxoplasma and Neospora infection which was negative (n = 5). CSF samples from the IS group were taken from either the cerebellomedullary cistern or lumbar subarachnoidal space and showed mixed neutrophilic pleocytosis with moderate to severe eosinophilia.Spirocerca Lupi nematodes. Four dogs improved clinically under treatment, two deteriorated progressively and the owners opted for euthanasia. At postmortem examination a Spirocerca Lupi nematode was identified in the caudal lumbar segments of the spinal cord and a granuloma was found in the esophagus. Outcome of 1 dog is missing.Clinical diagnosis was further supported by improvement under treatment or at postmortem examination with identification of The healthy group was represented by 6 neutered-male Beagles, 9 months old, clinical and neurological examinations revealed no pathological findings. CSF was obtained from the cerebellomedullary cistern in these dogs.Levels of AEA in CSF were found in the picomolar range (pM), in serum in the nanomolar range (nM). Concentrations of total AG in serum and CSF were expressed in the nanomolar range (nM).The type 3 test of fixed effects denies the hypothesis of equal means of AEA for all group levels in CSF  and serum .The highest values of AEA in CSF were found in dogs with Intraspinal Spirocercosis and differed significantly with all other groups SRMA A (p = 0.0084), SRMA Tr (p<0.0001) and healthy dogs (p = 0.0001). Levels of AEA in CSF were significantly increased in SRMA A compared to SRMA Tr (p = 0.0482). Furthermore, healthy controls showed significantly lower concentrations of AEA in serum in comparison to dogs with Intraspinal Spirocercosis (p = 0.0032), SRMA A (p = 0.0111), and SRMA Tr (p = 0.0029).Medians and ranges of AEA concentrations in CSF and serum are summarized in The type 3 test of fixed effects denies the hypothesis of equal means of total AG for all group levels in CSF  and serum .Levels of Total AG in CSF were significantly increased in Intraspinal Spirocercosis compared to SRMA A (p = 0.0072), SRMA Tr (p< 0.0001) and healthy dogs (p = 0.0005). Furthermore, statistically significant differences were found between SRMA A and SRMA Tr, (p = 0.0016). Moreover, dogs suffering from SRMA in the acute phase and those with Intraspinal Spirocercosis showed significantly higher concentrations of total AG than healthy controls in serum .Medians and ranges of total AG concentrations in CSF and serum are summarized in Cannabinoid receptor type 2 immunoreaction was detected in spleen and liver samples of healthy dogs serving as positive controls. Moderate to strong immunoreaction was detected in numerous cells of the red pulp, as well as in the periarteriolar lymphoid sheath (PALS), marginal and mantel zone of splenic follicles (white pulp), while only few positive cells were found in the germinal center of splenic follicles . HepatocIn spinal cord sections of healthy control dogs, CB2 was moderately to strongly expressed in numerous small vesicles in the cell membrane by approximately 90% of glial cells which were homogeneously distributed throughout the grey and white matter . Moreovei.e. lymphocytes, plasma cells, neutrophils, and macrophages). In most dogs, inflammatory infiltrates were found within the adventitia of vessels, perivascular, subdural, and subarachnoidal, but in few dogs inflammatory cells were found within the white and grey matter  compared to healthy controls. Moreover, dogs suffering from SRMA in the acute phase and those with Intraspinal Spirocercosis showed significantly higher concentrations of total AG than healthy controls in serum. SRMA is a systemic immune-mediated disorder . Even whStrong CB2 immunoreaction was found in leukocytes within the marginal zone of splenic follicles and hepatocytes and Kupffer cells as previously described , 73, 74.i.e. neutrophils, lymphocytes, plasma cells, eosinophils and macrophages) in spinal cord lesions of dogs with SRMA and IS. Interestingly, arachnoid mesothelial-like cells and cells in the pia matter near the lesions expressed also CB2. Immune cells express high levels of CB2, and even a hierarchy of CB2 receptor expression has been described within the immune system  [CB2 was strongly expressed on infiltrating leukocytes (hocytes) . The levhocytes) . CB2 rechocytes) , 89.Endocannabinoids have been shown to downregulate inflammation in numerous experimental models –26. Actiin vitro and in vivo experiments have reported a pro-inflammatory role of the endocannabinoid system in the development of inflammation [Although several studies support endocannabinoids as anti-inflammatory mediators, several ammation . These pammation , 95, 96.ammation . Indeed,ammation . The antThe endocannabinoid system response in inflammation is indeed dependent on type and state of the disease. While CB2 receptor activation in general mediates immunosuppressive effects limiting inflammation and being associated with tissue damage in several pathological conditions, in some disease states activation of the CB2 receptor may enhance or even trigger tissue damage . In physAlthough the initial enhancement of the endocannabinoid system has been proven to have an endogenous protective response and attempts to control inflammation, as the disease progresses, a later dysregulation of the endocannabinoid system during neuroinflammation leads to increased CB2 receptor expression, excessive endocannabinoid production leading to an exacerbated inflammatory response. Thus, both CB2 agonists and antagonists might be beneficial in counteracting the inflammatory consequences depending on the disease phase . IncreasThe present study revealed an upregulated endocannabinoid system in canines with inflammatory CNS diseases, highlighting the endocannabinoid system as a potential target for treatment of inflammatory CNS diseases. Levels of ECs in CSF of dogs with inflammatory CNS diseases were increased compared with healthy dogs, and those under treatment. Furthermore, dogs with eosinophilic pleocytosis showed higher level of ECs than those with neutrophilic pleocytosis. Such pleocytosis could be due to the effects of ECs on eosinophil migration to the CSF. We also found upregulated expression of CB2 receptors in SRMA and IS lesions. Such an upregulation of AEA and 2-AG together with an overexpression of CB2 receptors may indicate a dysregulation of the endocannabinoid system and may be involved in the inflammatory response. Therefore, the development of new anti-inflammatory treatment strategies in canine CNS inflammation should involve the EC system.


Cryptococcus yeasts can be distinguished using trypan blue staining. We hypothesized that trypan blue staining combined with haemocytometer counting may provide a rapid estimation of quantitative culture count and detection of CSF sterility. To test this, we evaluated 194 CSF specimens from 96 HIV-infected participants with cryptococcal meningitis in Kampala, Uganda. Cryptococcal meningitis was diagnosed by CSF cryptococcal antigen (CRAG). We stained CSF with trypan blue and quantified yeasts using a haemocytometer. We compared the haemocytometer readings versus quantitative Cryptococcus CSF cultures.Quantitative culture is the most common method to determine the fungal burden and sterility of cerebrospinal fluid (CSF) among persons with cryptococcal meningitis. A major drawback of cultures is a long turnaround-time. Recent evidence demonstrates that live and dead 10 transformed counts were 5.59  for haemocytometer and 4.98  for culture; while the overall median counts were 5.35  for haemocytometer and 3.99  for cultures. The percentage agreement with culture sterility was 2.4% (1/42). Counts among non-sterile follow-up specimens had a median of 5.38  for haemocytometer and 2.89  for culture. At diagnosis, CSF quantitative cultures correlated with haemocytometer counts . At 7–14 days, quantitative cultures did not correlate with haemocytometer counts .Haemocytometer counting with trypan blue staining had a sensitivity of 98% (64/65), while CSF cultures had a sensitivity of 95% (62/65) with reference to CSF CRAG for diagnostic CSF specimens. For samples that were positive in both tests, the haemocytometer had higher readings compared to culture. For diagnostic specimens, the median of logDespite a positive correlation, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative cultures in patients receiving antifungal therapy.The online version of this article (doi:10.1186/s12866-017-1093-4) contains supplementary material, which is available to authorized users. Cryptococcus yeasts in CSF , wit, wit8], Cryptococcus cells, heat-killed Cryptococcus cells were stained with trypan blue and analysed by microscopy. All the heat-killed Cryptococcus cells took up the trypan blue stain , only one had a zero reading with the haemocytometer and the rest (n = 41) ranged in 5000 to 3,405,000 cells/ml. In other words, haemocytometer counting with trypan blue staining for viable Cryptococcus had excellent overall sensitivity of 97% but only agreed in 2% (1/42) of sterile cultures (McNemar’s P < 0.001). The positive predictive value for haemocytometer with reference to culture was 78% (147/189) while the negative predictive value (the probability that a zero reading by haemocytometer would have a negative culture) was only 20% (1/5).Using CSF CRAG as a diagnostic reference, trypan blue staining with haemocytometer counting had a sensitivity of 98% (64/65) while CSF cultures had a sensitivity of 95% (62/65) for diagnostic CSF specimens. Among CSF samples collected longitudinally during antifungal treatment , the haemocytometer had more positive readings (97% [189/194]) than quantitative fungal culture (78% [151/194]) with reference to CSF CRAG. Among samples with no culture growth (10-transformed live cells/ml) to quantitative fungal cultures (log10-transformed CFU/ml), culture consistently had lower counts than haemocytometer. The overall median counts were 5.35 log10 yeasts/mL  for haemocytometer counting of trypan blue stained cells and 3.99 log10 CFU/mL  for quantitative cultures. For diagnostic specimens only, the median of log10 transformed counts were 5.59  for haemocytometer and 4.98  for culture. Among 151 cultures with growth, there was a significant difference in the means of counts between culture and haemocytometer (p < 0.001); though there was a significant relationship between haemocytometer and culture overall by linear regression .When comparing the readings of trypan blue stain using a haemocytometer  and day 3   or day 14  of amphotericin B therapy. There was no significant relationship between the haemocytometer counts with trypan blue and baseline opening pressures . However, there was a significant correlation between culture and opening pressures .There was a significant correlation between the haemocytometer and culture readings at day 1  between the two methods at day 1 and 3.Despite a significant relationship between the two methods and a positive correlation in the readings during the first week of treatment, the haemocytometer counts with trypan blue staining did not predict the outcome of quantitative culture, and culture had significantly lower values than the haemocytometer. However, linear regression showed a significant relationship  between the two counting methods which became more pronounced with time on antifungal therapy. Furthermore, trypan blue staining was completely unreliable at predicting conversion to culture sterility. The clinical implication given these results is that staining with trypan blue cannot be used as a rapid surrogate for CSF cultures to help determine duration of therapy or early discontinuation of amphotericin in cryptococcal meningitis. Whether there could still be a role for trypan blue or other vital staining techniques to distinguish between culture-confirmed relapse and immune reconstitution inflammatory syndrome (IRIS) with sterile CSF is unknown. It is possible that trypan blue uptake could be more complete in yeast cells that have been dead for longer periods of time and have not yet been cleared from CSF, as has been observed in IRIS using traditional staining techniques. We tested this by treating one cryptococcal isolate with amphotericin B for 24 h to kill the yeasts with an antifungal agent. The isolate treated with amphotericin B, when mixed with trypan blue and observed under a light microscope, showed that 98% of the yeast cells were live and not killed by the antifungal treatment after 24 h.For both haemocytometer counts and culture, the fungal burden decreased over time while receiving amphotericin-based combination therapy. However, we observed significant differences (A recent study done by Bio-Rad Laboratories using HeLa cells compared the haemocytometer to the TC20 automated counter. Results revealed that automated cell counting could significantly reduce both user and concentration-dependent variances while reducing the turn-around-time . WhetherCryptococcus yeasts in the CSF.In conclusion, trypan blue staining using manual platforms was not helpful in the rapid detection of sterility of CSF and viability of