The measurement of head circumference (HC) is widely used in clinical and research settings as a proxy of neural growth. Although it could aid data collection, no studies have explored either the reliability of adult self-measurements or parental measurements of young children. This study therefore aimed to examine whether adult self and parental measurement of HC constitute reliable data.A total of 57 adults  were asked to measure their HC twice following written instructions (adult self-measurement). These measures were compared to those of a researcher independently measuring the same participant’s HC twice. Additionally, mothers of 25 children  were also asked to measure their child’s HC , and again this was compared to researcher measurements of the child’s HC. The intraclass correlation coefficient between adult self- and researcher measurement was 0.84 and between parent and researcher measurement was 0.99. The technical error of measurement was also acceptable, within the range of a skilled anthropometrist.The high degree of agreement between researcher and adult self-measurement/parental measurement of HC demonstrates that these different assessors produce similarly reliable and reproducible data. This suggests adult self- and parental measurements can reliably be used for data collection to enable valid large-scale developmental and clinical studies of HC. Head circumference (HC) is a widely used proxy of neural growth and brain size in paediatric and research settings . HC, as Larger samples and longitudinal data would enable a better understanding of this phenomenon. As HC is a relatively quick and cost-effective assessment and can provide information on risk for some medical and neurological problems, it would be useful to establish if the general population is as accurate as trained professionals in producing these measurements. Indeed, HC measurements are often considered difficult even by professionals due to individual differences in head shape, hair styles and texture, and subject cooperation as well as examiner differences in tape measure placement and tautness. Moreover, the authors are unaware of standardized guidelines for measuring HC and different agencies recommend different tape measure tools and techniques , althouIn total, 57 adults  in the Cambridge area were targeted via an opportunistic sampling method. A second opportunistic sample included 25 mothers of typically developing children  who were asked to measure their child’s HC.Participants were given a measuring tape and written instructions/diagram explaining the HC measurement: the instructions included a photograph of an adult male measuring his own HC, and asked the participant to measure their head above the ears and slightly above the eyebrows in order to capture the maximum distance around their head, and to pull the tape measure tightly. It was also emphasized that HC differences between individuals are small and that accuracy and detailed measurements  were essential  the adult-self or parental-child and the primary researcher, and (b) researcher 1 and researcher 2 measuring the adult’s HC.Separate absolute and relative TEM was calculated for the differences in the two HC measurements for the participant’s adult self- or parental measurements , researcher A , and researcher B (n = 47 adults). All relative TEM were below 1.0 and within the acceptable range for a skilled anthropometrist, with the TEM for the mothers measuring their child’s HC being especially low in error.Adult self-measurement intra-rater reliability, according to ICC calculations, was high, with the participants showing an ICC = 0.94,  0.90–0.96) between their two self-measurements. Researchers showed similarly high self-reproducibility, between their own two measurements of adults: Researcher A, ICC = 0.98 (95% CI: 0.97–0.99); Researcher B, ICC = 0.98 (95% CI: 0.96–0.99). The researchers measuring the children’s heads were also very reliable, with an ICC = 0.99  between their two measurements. Parental measures of their child’s head were very reliable . The 95% CI for the researcher ICC and the participant ICC overlapped; consequently, reliability estimates could not be statistically differentiated. In other words, self-measurements were as reliable as researcher measurements.To calculate between-rater TEM, two analyses were conducted. The first may be considered to be an index of self-/parent-measurement validity as the difference between the mean of the two self-/parent-measurements were compared with the mean of researcher A’s two measurements. Again, the between-rater relative TEM was in the acceptable range for a skilled anthropometrist for both adult self- and parental measurements  is comparable to previous studies looking at the reliability of adult self-measurements of other body parts  and the Adult self- and parental measurements of HC showed strong concordance with researcher measurements and the TEM was acceptable, suggesting little variability between the measurement of HC by the adult/parent and that of the trained researcher. Indeed the reliability of adult or parent measurements, according to the ICC 95% CIs, could not be statistically differentiated from those of researchers, suggesting that self-/parent measurements of HC reflect reliable data for both research and clinical purposes. These findings are comparable to those found for self-measurement of other body parts  and pareIt should be noted that, with regards to limitations, adults and children in this study were typically developing, so it is unclear whether these same levels of reliability would be found in clinical populations. Furthermore, it is recommended that researchers and professionals think carefully about their instructions and training if they intend to collect self-/parental HC measurements and to pilot test their approach before employing it in a large scale.To summarize, the results of this study provide strong evidence for confidence in adult self-measurements and parental measurements of HC, suggesting that trained researchers or clinicians may not always be required to obtain reliable HC measurements. This finding is applicable in paediatric, neurological, psychiatric, and psychological research, where more larger and rigorous studies might be conducted using lay measurements of HC, as well as in clinical practice.HC: Head circumference; ICC: Intraclass correlation coefficient; CI: Confidence interval; TEM: Technical error or measurement.The authors declare that they have no competing interests.JS, TT, AH, and SBC contributed to study design, JS, TT, and KA collected the data, JS and TT conducted statistical analysis, JS drafted the manuscript. All authors read and approved the final manuscript.Head circumference instructions.Click here for file


Drosophila (dKCNQ) has similar electrophysiological properties to neuronal KCNQ2/3, including conserved acute sensitivity to ethanol block, with the fly channel (IC50 = 19.8 mM) being more sensitive than its mammalian ortholog (IC50 = 42.1 mM). This suggests that the role of KCNQ in alcohol behaviour can be determined for the first time by using Drosophila. We present evidence that loss of KCNQ function in Drosophila increased sensitivity and tolerance to the sedative effects of ethanol. Acute activation of dopaminergic neurons by heat-activated TRP channel or KCNQ-RNAi expression produced ethanol hypersensitivity, suggesting that both act via a common mechanism involving membrane depolarisation and increased dopamine signalling leading to ethanol sedation.In humans, KCNQ2/3 channels form an M-current that regulates neuronal excitability, with mutations in these channels causing benign neonatal familial convulsions. The M-current is important in mechanisms of neural plasticity underlying associative memory and in the response to ethanol, with KCNQ controlling the release of dopamine after ethanol exposure. We show that dKCNQ is broadly expressed in the nervous system, with targeted reduction in neuronal KCNQ increasing neural excitability and KCNQ overexpression decreasing excitability and calcium signalling, consistent with KCNQ regulating the resting membrane potential and neural release as in mammalian neurons. We show that the single KCNQ channel in  Drosophila, which typically contains a single member of each Kv channel subfamily, with nulls being adult viable; this allows exploration of the functional consequence of complete lack of a subfamily of Kv channel Voltage-gated potassium (Kv) channels form a diverse gene family that, in humans, is subdivided into 12 subfamilies of 40 members KCNQ1 mutations cause Long and Short QT KCNQ1 mutations also result in adult onset type II diabetes KCNQ2/3 loss-of-function mutations result in a form of epilepsy. KCNQ4 loss-of-function mutations cause autosomal dominant deafness. M-current inhibitors increase excitability and have shown some promise in enhancing memory in models of dementia. Conversely, M-current openers are of great interest as anticonvulsants, analgesics and treatments of psychiatric diseases KCNQ (Kv7) channels mediate a range of important physiological functions, form a hotspot of genetic diseases and are targets for new and existing drug treatments. In human cardiac muscle, Drosophila has a single KCNQ channel (dKCNQ) that is most highly expressed in the nervous system dKCNQ loss-of-function mutations resulting in heart arrhythmia in young flies. This phenotype is also observed in aged wildtype flies, which correlates with an age-dependent decline in dKCNQ expression Drosophila is a powerful model of the molecular and neuronal mechanisms of alcohol and other addictive drug-related behaviours, with many of these genes and mechanisms identified in Drosophila and then validated in mammals Drosophila and rat KCNQ2/3 channels are acutely sensitive to block by low concentrations of ethanol. We characterise for the first time the in vivo consequence of dKCNQ mutations on neural activity and behaviour, showing a role for the channel in regulation of ethanol sensitivity and tolerance.Drosophila KCNQ RE26469 cDNA , rat KCNQ2 cDNA  and rat KCNQ3 cDNA  Drosophila RE26469 full-length KCNQ cDNA using the WU-BLAST server at EMBL-EBI.cDNAs were expressed in Human Embryonic Kidney (HEK293) cells using previously published protocols 0.5 value was the voltage required for half the maximal activation current. Mean V0.5 values are shown ± SEM. Data were analysed using Graphpad Prism.Whole-cell voltage-clamp recordings were made from HEK293 cells using an Axopatch 200A amplifier  as previously described KCNQ deletion mutant contains an imprecise excision of the EP2074 element (186KCNQ) that removes all the 5′ and transmembrane regions of the channel and therefore is likely a null mutation KCNQ control was a precise excision of the element (97KCNQ), leaving the gene completely intact uas-KCNQ flies allowed Gal4 promoter-driven overexpression of KCNQuas-KCNQ-RNAi (Bloomington stock 27252) allowed Gal4-targeted knockdown of the channel. Wild-type flies were Canton S w- (CSw-) from a stock previously maintained in the Griffith lab. All KCNQ mutant, Gal4 and uas lines were out-crossed with this CSw- line prior to behavioural analysis. All genotypes and all other crosses were raised on corn-meal malt-molasses agar medium at 22±2°C and 60±10% humidity under a 12∶12 h light-dark cycle.The 3, 115 mM sucrose, 4 mM MgCl2 5 mM trehalose, 1.5 mM CaCl2, and 5 mM HEPES, pH 7.3), and isolated brains from either stage were fixed in 4% paraformaldehyde in HL3.1 for 30 min before being washed in HL3.1 vGlut(OK371)-Gal4 pattern, a (1∶1000) rabbit anti-Drosophila Glut antibody was used KCNQ expression pattern was determined by visualising membrane-targeted GFP expressed using KCNQ-Gal4 reporter lines .Adult fly brains or third-instar larvae were dissected in HL3.1  for each time point. Images were processed using Volocity (PerkinElmer) and Image J (NIH). Data were statistically analysed and presented with Graphpad Prism software.Third-instar larvae were dissected in haemolymph (HL3.1) solution containing 1.5 mM CaAll experiments were performed at 25°C and 70% humidity under white light. Twenty synchronised 3–5 day old male and female flies were used. One millilitre of 40% ethanol solution was added to an absorbent pad at the bottom of a sealed bottle. Active flies initially remained on the walls or up beneath the bottle lid. During the test period, a number of flies became anesthetised and were immobilised at the bottom of the bottle. These flies were counted at 5-min intervals over the 90-min ethanol exposure to measure sensitivity Ethanol content of flies of different genotypes was determined by exposing twenty active flies to ethanol vapour in a sealed bottle that an absorbent pad soaked in 1 ml of 40% ethanol. Flies were frozen in liquid nitrogen and stored at −80°C. Flies were homogenised with 500 µl of Tris-HCl (pH 7.5) and spun at 4°C for 20 min at maximum speed. The clear supernatant was recovered and used as the test sample. The alcohol reagent kit  was used to measure total ethanol content using the ADH-NADH reagent test Total RNA was isolated from adult fly heads using a Trizol solution (Invitrogen). An equal number of age-matched control flies were frozen in liquid nitrogen and decapitated by vortexing. The detached heads were collected and homogenised. Trizol was added directly to the homogenised heads and RNA was extracted by ethanol precipitation. RNA was DNase-treated  and reverse-transcribed using a first-strand complementary DNA (cDNA) synthesis kit . The first strand cDNA was obtained from a total RNA template in a single reaction, adding a reverse transcriptase enzyme and oligo (dT) primers and incubating for 1 h at 42°C 5′-AGGAAAGCCGCTGAACTACA-3′, position 210–230,KCNQ forward: 5′-CGAGGTGCCCATTCCTAATA-3′, position 604–584.KCNQ reverse: The PCR amplification product was analysed by electrophoresis in 1% agarose gels and visualised by ethidium bromide staining and transillumination under UV light.Gal4 enhancer trap within the KCNQ gene was used to express GFP. These experiments revealed that KCNQ continues to be broadly expressed in the nervous system, including in glutamatergic motor neurons -Gal4 line that has particularly strong motor neuron expression in larvae. These neurons also express KCNQ . To explore how the level of KCNQ may be affecting neural excitability, we performed in vivo Ca2+ imaging in Drosophila. We used vGlut(OK371)-Gal4 to express a Ca2+ reporter, GCaMP3 2+ signalling that travels through the ventral ganglion. We measured the maximum change in fluorescence in the motor neuron soma when this occurs. Expression of KCNQ-RNAi caused a significant increase in neuronal excitability  neurons resulted in a wing-expansion phenotype  show an increase in sedation over 90 min. A KCNQ loss-of-function P-element mutant with greatly reduced KCNQ -Gal4Trh- and Tph-Gal4, Prior work has shown that aminergic (dopamine and serotonin) neurons mediate the neural response to addictive drugs such as ethanol sitivity ; conversTRPA1, a channel known to cause a large depolarisation of Drosophila neuronal membrane potential that triggers firing action potentials without substantial inactivation of Na+ channels KCNQ-RNAi in neurons. Heat activation of Th-Gal4, uas-TRPA1 showed an ethanol hypersensitivity phenotype but did not cause any other non-specific locomotor defect .KCNQ expression decreases in aged flies , with thA receptors + channels but closes Shaw K+ channels Drosophila KCNQ channel is more sensitive to ethanol than the mammalian channel, with an IC50 of 19.8 mM compared to 42.1 mM for KCNQ2/3  and KCNQ deletion mutants hybridised with the sense probe (D.) showed little or no non-specific staining.(PDF)Click here for additional data file.Figure S2Changes in DDC neuron KCNQ levels leads to a wing expansion defect associated with impaired release A. Histogram showing that increasing or decreasing the level of KCNQ in Ddc neurons results in an increase (p<0.01) in the frequency of the wing expansion defect compared to control . Data were analysed by 1-way ANOVA with a Bonferroni post-hoc test .(PDF)Click here for additional data file.Figure S3Ethanol does not cause a change in voltage-dependent activation of mammalian or Drosophila KCNQ channels. The G–V relation for KCNQ2/KCNQ3 (A.) shows no apparent shift (p>0.05) by 10 mM ethanol , compared to control . B. The dKCNQ control current  and the current in the presence of 10 mM ethanol  show that the blocker does not cause a significant (p>0.05) shift in whole cell current activation. Activation relations were calculated from tail currents. Data were analysed by Student's paired t-test. n≥4. For all figures: error bars are standard error of the mean and no asterisk means not significant; *p<0.05, **p<0.01 and ***p<0.001.(PDF)Click here for additional data file.Figure S4KCNQ signalling does not affect ethanol metabolism or avoidance.A. The sedation assay was performed in the absence of ethanol in order to control for any possible non-specific locomotor effect caused by heating flies. In the absence of ethanol, flies do not become sedated, so the per cent sedated or T50% sedated is not possible to calculate. Instead, the number of flies at the bottom of the bottle at a given time was counted. Th-Gal4, + , Th-Gal4, + , Th-Gal4, uas-TRPA1  and Th-Gal4, uas-TRPA1  were compared . B. Heat activation of TRPA1 in Th neurons did not predispose flies to sedation or cause a non-specific locomotor deficit. The number of Th-Gal4, + and Th-Gal4, uas-TRPA1 flies at the bottom of the bottle was counted at 23 or 30°C  and 2-way ANOVA indicates that genotype and temperature did not affect this number (p>0.05). C. The ethanol content of experimental and control (CSw- wild-type and KCNQ control) genotypes were quantified using an alcohol-dehydrogenase-based assay, in which the absorption levels of all genotypes were similar (p>0.05) at the end of the 90 min exposure to 40% ethanol vapour. Likewise, the rate of catabolism as reflected by the ethanol content after 50 min recovery from the exposure was the same between genotypes. D. Experimental and control  flies similarly (p>0.05) avoided the arm of the T-maze containing 40% ethanol. All data were analysed by 1-way ANOVA with Bonferroni post-hoc test.(PDF)Click here for additional data file.


Background. Organ shortage has liberalised the acceptance criteria of grafts for heart transplantation, but which donor characteristics ultimately influence the decision to perform transplantation? For the first time this was evaluated using real-time donor data from the German organ procurement organization (DSO). Observed associations are discussed with regard to international recommendations and guidelines. Methods. 5291 German donors (2006–2010) were formally eligible for heart donation. In logistic regression models 160 donor parameters were evaluated to assess their influence on using grafts for transplantation . Results. Successful procurement was determined by low donor age , large donor height , exclusion of impaired left ventricular function or wall motion , arrhythmia , and of severe coronary artery disease . Donor characteristics differed between cases where the procedure was aborted without and with allocation initiated via Eurotransplant.  According to the International Society for Heart and Lung Transplantation (ISHLT), worldwide about 5000 heart transplants are carried out yearly . In GermThis study evaluates for the first time which factors ultimately influence the decision to use a graft for heart transplantation or not by using real-time data of donation procedures provided for allocation and decisions at recipient center level. The actual decision pathways in the donation process are discussed with reference to the published recommendations and guidelines. At different levels they are more or less triggered by the organ procurement organization (OPO) or recipient centers.Optimal evaluation of potential donor hearts is a cost-intensive purpose, including special cardiac diagnostic procedures like coronary angiography. Strategies to avoid a waste of resources have to be discussed in health policy.The national OPO, the German Foundation for Organ Transplantation (DSO), coordinates all organ donations in Germany. Only allocation is outsourced by law to the international organ exchange organization Eurotransplant (ET). In total, 15 484 pseudonymous DSO data records from potential or realised donors from January 2006 to December 2010 are available. Over 160 donor variables were analysed. All free text results of imaging examinations  were assessed by three experts in a review process; the results were classified according to the risk estimates published by Grauhan et al. on behalf of the national transplantation society (Deutsche Transplantationsgesellschaft (DTGs)) [n = 6376) were excluded. Records of donations from outside of Germany with grafts transplanted in the country had to be excluded because of lack of medical data as the DSO was not entitled to records beyond administrative issues (n = 1821). Cases were excluded if the entire donation process had been terminated before registration of the donor with ET for allocation of at least one organ, because in these cases only limited data about medical reasons were documented, which is in accordance with the rules of national personal data protection laws (n = 1996) (n = 3536), and a validation sample (n = 1755). First, the dependency of heart transplantation on each potentially influencing variable in the training sample was investigated using univariate analyses. We used the χ2 test, the Jonckheere Terpstra test, and the measure of association Gamma; quantitative characteristics were evaluated using empirical distribution curves and the Kruskal Wallis test.Because this paper aims to evaluate the medical reasons for deciding for or against realisation of heart transplantation from potential donors, all cases where heart donation was impossible due to legal reasons  . The remhttp://dx.doi.org/10.1155/2013/701854):inclusion criteria:medical relevance, frequency of quotation in the literature;P value of score test ≤0.2 (univariate logistic regression);OR, width of confidence interval, and exclusion criteria:high number of missing values;possible interactions and correlations with other variables ;surrogates of variables already included in the model;extremely rare comorbidities (positive findings <0.5%).The multivariate analysis was conducted using logistic regression. The following criteria were used to determine which of the 160 investigated variables were included in the multivariate analysis . To select explanatory variables during model building, forward stepwise selection was performed. To validate the results and as a sensitivity analysis, a backward selection was also applied. Model testing was done using the validation sample. To quantify the ability of the logistic regression model to predict results, the proportion of concordant pairs (pairs where observed and predicted results (heart transplantation yes/no) agree) was determined.  Level A: Registration of the donor with ET for allocation of at least one organ with the heart excluded for medical reasons based on data from the OPO.  Level B: Registration of the donor with ET for allocation including the heart, but heart transplantation not realised more or less based on decisions by recipient centers.  Level C: Registration of the donor with ET for allocation including the heart and heart transplantation realised.Three different decision levels exist regarding the realising of heart transplantation from a potential donor: The results of multivariate analysis comparing Level A to C and Level B to C separately are described. Data from the training sample are shown .n = 1425; 76.5%), as were the majority of the donors . The recipients were on average 7.5 years older than the donors. Up to 15 years of age donors and recipients were age-matched, probably due to size matching. Recipients older than 70 years received organs from elderly donors. Between these two margins, the donor and recipient ages were not well matched  and 96.6% (electrocardiography), respectively.P = 0.02; B: 1.08, 95% CI: 0.77–1.51, P = 0.67), type of brain damage , and length of time spent in the intensive care unit . Different decision patterns are described. On the one hand, there are the coordinators, who decide whether or not a potential donor should be allocated by ET (Level A), and on the other hand there are factors decisive for the final result (Level B) that exist between the start of allocation by ET and the heart surgeon's final judgment of the donor organ. Varying odds ratios of some influencing variables between Levels A and B  indicateP values. All the variables that were selected in the forward selection process in the training sample were remodeled using the validation sample. The results are shown in . All models showed a high congruence in the proportion of concordant pairs. In particular variables with a significant impact and not coding the category of missing values showed similar odds ratios and Recovery of a heart for heart transplantation was limited at Level A by donor age, height, gender, secondary brain lesions, length of stay in ICU, need for high inotropic support, severe functional abnormalities detected by ECG or echocardiography, and evidence of coronary heart disease . At LeveIn summary, donor age, body size, a coronary artery disease diagnosis, and the results of using the German Transplant Society's parameter catalog for judging donor hearts , 4 regardonor age is a known risk factor limiting survival after heart transplantation [P < 0.0001; for Level B: 0.95, 95% CI: 0.94–0.96, P < 0.0001). Greater donor size was related to a higher rate of grafts used for heart transplantation . This can be explained by the need to avoid heart transplantation with an undersized graft [High antation , 15. In ed graft .coronary heart disease (CHD) limits the use of grafts for heart transplantation [P < 0.0001), thereby virtually excluding transplantation. The ISHLT guideline recommends not using grafts with obstruction of the main coronary arteries [arterial hypertension was a significant risk factor at Level A . Generally arterial hypertension may result in left ventricular hypertrophy (LVH). Severe LVH detected by echocardiography was associated with an increased risk of not using the graft for heart transplantation at Level A  and Level B . According to the ISHLT, using grafts with LVH appears to be appropriate if an ECG shows no evidence of LVH, and the thickness of the left ventricular wall does not exceed 14 mm [heart valve morphology  were also a risk factor for not using the graft . According to the ISHLT, there is no reason to prevent the use of donor hearts with valvular insufficiencies, especially since intra-operative corrections are possible [wall motion abnormalities detected by echocardiography were a highly significant factor for excluding a heart from donation  when compared to normal function . Hearts with an insignificant reduction in LVF are used as frequently as hearts with normal LVF; this is not listed as a reason to exclude a donor heart in the existing guidelines. However, there is considerable hesitation to transplant hearts that have abnormal wall motion, such as regional akinesia or global hypokinesia. Whether these abnormal regional wall movements are correlated with consequential damage due to coronary sclerosis which entails irreversible damage or are the result of temporary neurocardiac dysfunction which entails reversible damage [infections like meningitis, pneumonia, cytomegalovirus, toxoplasmosis, and recovery from sepsis were insignificant risk factors in multivariate analysis. This is in agreement with the results of other authors [laboratory parameters, the heart enzymes are considered as relevant for donor heart selection [μg/L), Level A adjusted OR: 0.19, 95% CI: 0.07–0.54, P = 0.0018). This observation underlines Grauhan et al.'s hypothesis that ascertaining the Troponin level is of greater importance than the CK-MB levels, which can also be elevated due to cerebral damage [The diagnosis of antation . The adjarteries . But accarteries . Ultimatarteries . Of the possible , 19. Reldonation . The sam authors . Howevercatecholamines, the ISHLT recommends rejecting a potential donor heart if there is a great need for inotropic substances (dopamine 20 μg/kg/min or similar doses of other adrenergics except for forced optimisation of preload and afterload) [μg/kg/min were different between Levels A and C; however, this was not true for Levels B and C (Regarding the need for terload) . In the  B and C . This coLimitations of the current analysis include the fact that it concerns a secondary data analysis, where collection of the data variables is not tailored exactly to the research question , and theThe DSO data used in this analysis comprise a comprehensive data set that includes all potential donors in Germany during a time span of five years. The target population is therefore completely captured, and selection bias is limited to potential donors who were never registered in the DSO . A further strength of this study is the separation of the population into training and validation samples, so that results were checked within the same basic population. Between the training and validation samples, all models showed a high congruency in terms of the proportion of concordant pairs, and the different variable selection procedures (forward and backward) showed similar to identical results, thereby confirming the variables selected during model development based on the univariate results.Thus, the current analysis clearly demonstrates that both guidelines of the ISHLT and German national recommendations (DTG) are more or less implemented in daily practice of heart donor evaluation in Germany. This study also showed that, within the donation process different donor, characteristics determine successful recovery of a heart for transplantation and that the impact of specific donor variables determine the fate of a graft at different decision levels. This should be taken in account by the implementation of binding recommendations or guidelines. Suboptimal evaluation of potential donor hearts result in a waste of resources with needless binding of capacities already in the run-up to transplantation and disappointing results which are difficult to explain to recipients and their relatives. Hence, standardisation of recommendations of the evaluation procedures for potential donor hearts and their mandatory implementation should be an issue of health policy.The current analysis is the first and exhaustive review of donor heart allocation process including all potential donors in Germany during a time span of five years.International and national guidelines are more or less implemented in daily practice of heart donor evaluation in Germany, but the impact of specific donor variables determines the fate of a graft at different decision levels of the allocation process.For health policy, standardisation of the evaluation procedures for potential donor hearts and their mandatory implementation should be promoted.Additional Supporting Information may be found in the online version of this article.Table S1: List of variables included in the logistic regression model.Table S2: Multivariate logistic regression results from the training sample and model-checking on the basis of the validation sample (Level A versus Level C):High congruence in the proportion of concordant pairs.p-values. Changes from OR > 1 to OR < 1 or vice versa usually occurred in variables with wide confidence intervals and insignificant p-values, indicating that this might be due to random variation. This occurred in parameters as well as in categories representing missing values or results reported inadequately due to other more important pathologies (marked as “??”). Other categories of the same parameter showed congruent results of training sample and validation sample.The majority of variables in the models showed similar odds ratios and Table S3: Multivariate logistic regression results from the training sample and model-checking on the basis of the validation sample (Level B versus Level C):High congruence in the proportion of concordant pairs.p-values. Changes from OR > 1 to OR < 1 or vice versa were usually occurred in variables with wide confidence intervals and insignificant p-values, indicating that this might be due to random variation. This occurred in parameters as well as in categories representing missing values or results reported inadequately due to other more important pathologies (marked as “??”). Other categories of the same parameter showed congruent results of training sample and validation sample.The majority of variables in the models showed similar odds ratios and Table S4: Explanations and abbreviations in Tables S2 and S3.Click here for additional data file.


Guidelines for safe gamete donation have emphasised donor screening, although none exist specifically for testing oocyte recipients. Pre-treatment assessment of anonymous donor oocyte IVF treatment in Ireland must comply with the European Union Tissues and Cells Directive (Directive 2004/23/EC). To determine the effectiveness of this Directive when applied to anonymous oocyte recipients in IVF, we reviewed data derived from selected screening tests performed in this clinical setting.n = 225) in the anonymous oocyte donor IVF programme at an urban IVF referral centre during a 24-month period were analysed. Patient age at programme entry and clinical pregnancy rate were also tabulated. All recipients had at least one prior negative test for HIV, Hepatitis B/C, chlamydia, gonorrhoea and syphilis performed by her GP or other primary care provider before reproductive endocrinology consultation.Data from tests conducted at baseline for all women enrolling as recipients (Mean (±SD) age for donor egg IVF recipients was 40.7 ± 4.2 yrs. No baseline positive chlamydia, gonorrhoea or syphilis screening results were identified among recipients for anonymous oocyte donation IVF during the assessment interval. Mean pregnancy rate (per embryo transfer) in this group was 50.5%.When tests for HIV, Hepatitis B/C, chlamydia, gonorrhoea and syphilis already have been confirmed to be negative before starting the anonymous donor oocyte IVF sequence, additional (repeat) testing on the recipient contributes no new clinical information that would influence treatment in this setting. Patient safety does not appear to be enhanced by application of Directive 2004/23/EC to recipients of anonymous donor oocyte IVF treatment. Given the absence of evidence to quantify risk, this practice is difficult to justify when applied to this low-risk population. Since 2006, the European Union Tissues and Cells Directive (Directive 2004/23/EC) has exerted an important regulatory impact on the provision of clinical fertility practice in the E.U . This leStudies of screening test results have found the current testing approach has a very poor yield when applied to non-donor IVF cycles in Ireland . Indeed,n = 225) who enrolled in the anonymous donor oocyte IVF sequence at The Sims Institute (Dublin)/Sims IVF during a 24-month period ending January 2011. Because the study site is the only provider of anonymous donor oocyte IVF service in the Republic of Ireland, the geographical catchment area includes the entire state . All donor-egg IVF cycles reviewed in this study incorporated sperm provided by a known partner who was also separately evaluated, rather than from an anonymous sperm donor [N. gonorrhoea although this test was performed based on current ASRM testing guidelines for oocyte recipients in the United States [Written informed consent was obtained from all patients n = 22 who enroChlamydia organism which is present in the female reproductive tract, and has sufficient sensitivity to detect a single chlamydia cell in the sample submitted for analysis. The PCR test for C. trachomatis had no cross-reactivity with antigens from C. pneumoniae or C. psittaci (psittacosis), which require different detection methods.Testing was via PCR test performed either on cervical swab or random urine sample  . The tesN. gonorrhoeae DNA via spin-column chromatography and PCR amplification. The amplified PCR products were then further studied by agarose gel electrophoresis. This test incorporates reagents and enzymes for specific amplification of a 260 bp region of N. gonorrhoeae's PorA gene. A second heterologous amplification system is also used to identify possible PCR inhibition and/or inadequate isolation.Urine samples were used to detect bacterial T. pallidum. This protocol involves application of highly specific recombinant syphilis antigens for Abs that arise early during the primary stage of syphilis, and have lifelong positivity in most cases.Peripheral venipuncture was used to collect samples which were used with an enzyme immunoassay (EIA) method to detect IgM or IgG class antibodies (Abs) directed against Microparticle enzyme immunoassay (MEIA) techniques were used for qualitative detection of Hepatitis B surface antigen (HBsAg) in human serum as well as detection of antibodies to Hepatitis B core protein (anti-HBc). All samples were run on an AxSYM Plus diagnostic platform  utilizing microparticles coated with monoclonal antibodies for the detection of the above markers. Samples non-reactive by this testing modality are considered negative for Hepatitis B and need not be tested further. MEIA was also used for qualitative detection of antibody to hepatitis C virus (anti-HCV) in human serum. Similarly, simultaneous qualitative detection of antibodies to human immunodeficiency virus type 1 and/or type 2 (HIV-1/HIV-2) as well as the HIV p24 antigen in human serum was also performed. This global HIV assay (AxSYM HIV Ag/Ab Combo) does not, however, discriminate among HIV-1 antibody, HIV-2 antibody, or HIV p24 antigen reactivity. For these tests, assay result was determined by comparing fluorescent product formation rate to a cut-off rate, derived from previous AxSYM Index Calibrations. The sample is classified as reactive if the rate of formation of fluorescent product in specimens is greater than or equal to this threshold.In addition to results from the screening tests above, female patient age and clinical pregnancy rate (per embryo transfer) were recorded. Validation is required for any positive screening result, with reflex testing performed at the National Virus Reference Laboratory (UCD-Ireland).In the 24-month period ending January 2010, the mean (±SD) age for 225 consecutive patients who enrolled in anonymous donor oocyte IVF programme was 40.7 ± 4.2 yrs. At least one referring (primary care) physician had provided negative test results for HIV, Hepatitis B/C, chlamydia, gonorrhoea, and syphilis prior to reproductive endocrinology consultation and IVF .To comply with Directive 2004/23/EC as interpreted by Statutory Instrument (SI) 158 of 2006, SI 598 of 2007, a 2007 IMB Draft, and other IMB communications, HIV, Hepatitis B/C, chlamydia, gonorrhoea, and syphilis tests were again carried out at our centre and designated "baseline studies". All test results were reviewed first by clinic personnel with secondary verification by staff at the commercial laboratory responsible for sample processing. All specimens were marked as satisfactory for analysis and no positives were identified for any specimen submitted. Among patients undergoing anonymous donor oocyte IVF, the mean clinical pregnancy rate (per embryo transfer) during the study interval was 50.5%. All patients completed IVF without complications and there were no adverse events reported among recipients or anonymous oocyte donors.In recent years, mean age of infertility patients at initial consultation appears to have increased and has been accompanied by a sharp rise in the number of prior failed IVF cycles . This rei.e., anonymous donor oocyte recipients), these findings agree with general pre-fertility treatment screening data independently published from another leading Irish IVF centre [In Ireland, investigations focusing on screening data from non-donor IVF patients have likewise concluded that such individuals constitute a very low risk group, where the level of disease surveillance in this subgroup should be different compared to the general population. For Irish IVF patients and the physicians who offer this treatment here, the challenge to define proper testing is particularly acute given the absence of any legislation dealing with assisted reproductive techniques here . The curF centre  where ala priori evidence that "best practice standard" has been met. Continued use of Directive 2004/23/EC to justify repeat testing for HIV, Hepatitis B/C, syphilis, gonorrhoea, and chlamydia specifically among recipients of anonymous donor oocyte IVF treatment is unwarranted in the absence of medical evidence to quantify risks for this patient population.Making sure patients have no serious infection before commencing fertility treatment is important. But since patients in our study all had negative tests performed with their GP or other primary care provider before embarking on IVF treatment, our (repeat) screening provided no additional clinically relevant information. We can therefore endorse the general view that re-screening of IVF patients in Ireland is not an effective method to enhance patient safety . Indeed,The authors declare that they have no competing interests.APHW was the lead investigator and directed data aggregation, ABO, DJW and US were consultants who supervised patient screening and participated in data collection, KDM directed laboratory operations and ESS developed the project and coordinated the manuscripts. All authors read and approved the final manuscript.


Throughout the world there is an insufficient supply of donor organs to meet the demand for organ transplantations. This paper presents a protocol for a randomised controlled trial, testing whether a simple, theory-based anticipated regret manipulation leads to a significant increase in posthumous organ donor registrations.We will use a between-groups, prospective randomised controlled design. A random sample of 14,520 members of the adult Scottish general public will be contacted via post. These participants will be randomly allocated into 1 of the 4 conditions. The no questionnaire control (NQC) group will simply receive a letter and donor registration form. The questionnaire control (QC) arm will receive a questionnaire measuring their emotions and non-cognitive affective attitudes towards organ donation. The theory of planned behavior (TPB) group will complete the emotions and affective attitudes questionnaire plus additional items assessing their cognitive attitudes towards organ donation, perceived control over registration and how they think significant others view this action. Finally, the anticipated regret (AR) group will complete the same indices as the TPB group, plus two additional anticipated regret items. These items will assess the extent to which the participant anticipates regret for not registering as an organ donor in the near future. The outcome variable will be NHS Blood and Transplant verified registrations as an organ donor within 6 months of receiving our postal intervention.This study will assess whether simply asking people to reflect on the extent to which they may anticipate regret for not registering as an organ donor increases organ donor registration 6 months later. If successful, this simple and easy to administer theory-based intervention has the potential to save lives and money for the NHS by reducing the number of people receiving treatments such as dialysis. This intervention may also be incorporated into future organ donor campaigns.ISRCTN92204897ISRCTN:  There is an insufficient supply of donor organs to meet the demand for organ transplantations worldwide. Over 10,000 UK residents are currently on the waiting list for a solid organ transplant, and 3 patients die per day before they receive a transplant . DespiteTraditional social-cognitive models postulate that actions are determined by rational-cognitive factors. For example, the theory of planned behaviour (TPB)  suggestsAlthough the TPB has been found to predict a variety of actions ,6, incluanticipate the amount of regret that one would feel for undertaking or failing to perform an action, thereby giving people a pre-emptive strategy for avoiding this aversive emotion . We found that simply asking people whether they would later regret not registering as an organ donor increased their intentions to register . Moreover, we replicated this finding with a more representative sample of the adult Scottish general public . Although these findings are promising, people do not always act upon their intentions, the well recognised intention-behaviour gap. In a third pilot study we found that asking people whether they would later regret not registering as an organ donor increased self-reported organ donor registration . Her. Her39]]Plus-6  and StatPlus-6 . We willPlus.If our intervention is successful, we will test the processes through which it occurs. We will assess whether the anticipated regret intervention promotes organ donor registration by increasing people's intentions to register and decreasing their emotions and non-cognitive affective attitudes towards organ donation. Essentially, we will test whether intentions and emotions mediate the effect of our intervention on registration. Simple tests of mediation will be implemented in ZUMASTAT . More deThe effectiveness of the simple anticipated regret intervention will be assessed using the instrumental variables logistic regression analyses outlined above. This will determine whether there is a significant difference in the proportion of registered organ donors in each arm 6 months following our intervention. If we find that there is greater proportion of participants registered as organ donors in the AR arm than the other three arms, we will conclude that a simple anticipated regret intervention increases organ donor registration. We will also assess the mechanisms behind this intervention. The mediation analyses outlined above will test whether any significant effects of our intervention are due to increases in people's intentions to register and decreases in their emotions and non-cognitive affective attitudes towards organ donation.This study has received ethical approval from the South East Scotland Research Ethics Committee (ref: 11/SS/0093). This is an 18-month project. Months 1-4 will be spent designing, piloting and finalising the layout of the questionnaire pamphlet, and also in working closely with Perspektiv in randomising participants to the four arms of the study. The questionnaire packs will be posted out to participants in months 5 and 6. During months 7-12 the responses will be entered into the PASW spreadsheet as they are returned, and the analytic strategy will be finalised. The protocol for linking the participants to the NHSBT Organ Donor Register will also be developed and finalised during this period. During months 13 & 14 the NHSBT Organ Donor Register will be checked for new registrations by our participants. Months 15-16 will be spent conducting the final analyses, and in months 17-18 we will draft scientific papers, conference presentations and the Chief Scientist Office final report.A recent report by Nuffield Council on Bioethics  highlighNHSBT verified organ donor registration. Our proposed intervention is both simple and easy to administer, thereby making it a feasible strategy for improving organ donor registration. If this intervention is successful, we will liaise with our NHS partner (NHSBT) to discuss how this intervention could potentially be used in organ donor campaigns. This research, therefore, has the potential to dramatically increase the number of people who are registered as posthumous organ donors. This will save the lives of people who currently need a transplant and those who may need a transplant in the future. Moreover, with the average cost of dialysis at £30,800 per patient per year [Previous research has found that a simple, theory-based anticipated regret manipulation increases people willingness to register as an organ donor  and selfper year , this reThe authors declare that they have no competing interests.REO, EF and PCH obtained a grant to fund this project from the Chief Scientist Office (Scottish Government). LS drafted this protocol paper. REO, EF and PCH critically examined the paper. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/169/prepub


Meningeal inflammation, including the presence of semi-organized tertiary lymphoid tissue, has been associated with cortical pathology at autopsy in secondary progressive multiple sclerosis (SPMS).  Accessible and robust biochemical markers of cortical inflammation for use in SPMS clinical trials are needed.  Increased levels of chemokines in the cerebrospinal fluid (CSF) can report on inflammatory processes occurring in the cerebral cortex of MS patients.  A multiplexed chemokine array that included BAFF, a high sensitivity CXCL13 assay and composite chemokine scores were developed to explore differences in lymphoid  and inflammatory  chemokines in a small pilot study.  Paired CSF and serum samples were obtained from healthy controls (n=12), relapsing-remitting MS (RRMS) (n=21) and SPMS (N=12). A subset of the RRMS patients (n = 9) was assessed upon disease exacerbation and 1 month later following iv methylprednisone. SPMS patients were sampled twice to ascertain stability. Both lymphoid and inflammatory chemokines were elevated in RRMS and SPMS with the highest levels found in the active RRMS group. Inflammatory and lymphoid chemokine signatures were defined and generally correlated with each other. This small exploratory clinical study shows the feasibility of measuring complex and potentially more robust chemokine signatures in the CSF of MS patients during clinical trials. No differences were found between stable RRMS and SPMS. Future trials with larger patient cohorts with this chemokine array are needed to further characterize the differences, or the lack thereof, between stable RRMS and SPMS.    The pathology that drives progressive forms of MS remains poorly understood. SPMS was viewed traditionally as primarily a neurodegenerative process, yet recent studies indicate that neurodegeneration in SPMS is secondary to inflammation –4. ExtenIt remains unclear if there are fundamentally different inflammatory pathologies leading to the various lesions in the subpial and cortical gray matter relative to the white matter in MS as well as what is the relative contribution of the cortical component to progressive MS. Likewise, it is uncertain whether the TLT-type CNS events are particularly pathogenic by providing nurturing and stable B-cell niches in the meninges surrounding the brain and spinal cord, although a recent rodent EAE study suggested TLT were critical for determinant spreading in T cells –12. In MS, inflammation is associated with altered patterns of chemokine and cytokine expression . In geneIt is important to understand if RRMS and SPMS have different chemokine signatures in either CSF or the blood. Robust analytical methods are needed to measure longitudinal changes in the levels of these chemokines that can be present at rather low levels. In this manuscript, we explore the utility of quantitative panels of inflammatory chemokines, i.e. CCL2, CXCL9, CXCL10 and CXCL11 as well as lymphoid chemokines, CXCL12, CXCL13, CCL19 and CCL21, for use in blood and CSF. The methodology was rigorously validated, and an additional high sensitivity assay was developed for CXCL13 in CSF to adequately address the shortcoming of the existing method. Serum and CSF samples were collected at the Multiple Sclerosis Center of Northeastern New York  from 12 clinically stable RRMS (RRMS) patients who did not have any relapses or steroid treatment for at least 60 days; nine RRMS patients with an acute exacerbation by Schumacher’s criteria (EX-RRMS); 12 SPMS patients diagnosed on the basis of accumulation of disability independently of relapses and without evidence of ongoing relapses and/or steroid treatment for at least 90 days; and 12 healthy volunteers as normal controls (NC). All nine EX-RRMS subjects were seen within 48 hours of calling the MS Center with new onset symptoms of fewer than 6 days and clinically evaluated for objective findings. They underwent lumbar puncture (LP) the same day with blood drawn within 20 minutes of the LP. The first dose of 1000 mg of IVMP was begun within one hour of the LP for the first of three consecutive days. Eight of 9 EX-RRMS subjects received a brain MRI within 24 hours of the LP. One month after IVMP, these patients had a second LP. SPMS subjects underwent a second LP within 1-18 months after the first LP. Blood and CSF were obtained from MS patients with approval of the BioMed Institutional Review Board for the Multiple Sclerosis Center of NE New York. All patients signed informed consent.When lumbar punctures were performed, venous blood samples were collected into serum separator tubes. Following centrifugation, the serum was stored frozen at -80°C until analysis. The CSF was centrifuged immediately at 200g for 10 minutes at 2-8°C, and the supernatant was removed, aliquotted into Eppendorf tubes and immediately stored at -80°C. Contamination of the CSF by blood was carefully assessed by multiple criteria including gross appearance, RBC counts, presence of hemoglobin or Defensin-A3 (neutrophil) RNA transcripts and Affymetrix hemoglobin and neutrophil RNA signatures . All chemokine assays including CXCL13 immuno PCR (i-PCR), and multiplex assays were qualified using fit-for-purpose biomarker assay qualification to assure assay performance . In prelR&D Systems MAb 801) conjugated to DNA (mAb-DNA) was prepared by chemical cross-linking, and purified by preparative chromatograpy . Capture antibody  was coated onto TopYield modules (Nunc), blocked to minimize non-specific binding, and then incubated overnight at 4° C with 1:1 mixture of CSF sample and the DNA-tagged anti-CXCL13 mAb. After incubation, modules were washed and PCR master mix added to each well. The modules were sealed and placed in a real-time PCR cycler. The resulting delta Ct values were plotted against CXCL13 concentration. A standard curve was constructed from the obtained delta Ct values using a four-parameter curve fit model and the concentration of CXCL13 in each sample was extrapolated from the curve. Monoclonal anti-human CXCL13 antibody  and biotin (detection reagent). Each capture:detection antibody pair was screened by a single Luminex immunoassay using recombinant protein as the standard and human serum or CSF as assay matrix. The final antibody pairs were selected based on the sensitivity, specificity, and the ability to detect individual analytes at biologically relevant levels. Performance of each assay was then further optimized as a part of a multiplex to ensure minimal cross reactivity while maintaining the sensitivity toward individual analytes. Blinded samples  previously tested in individual ELISAs were analyzed in the multiplex assay to determine the correlation of the results obtained in the multiplex assay compared to those obtained in each individual ELISA. The lower limit of quantification (LLOQ) for all the chemokines tested in serum and CSF matrices are shown in th percentile of all patient data was the upper limit and levels higher than that were assigned the value 1. With a composite score derived from three chemokines, the maximum value was 3. All composite scores were then transformed to a 100 point scale. With the serum chemokine data, there was little movement and hence a composite scale as described above was inappropriate. Therefore, individual chemokine levels were converted to z-values and the composite scores represent the mean of the individual z-values. Statistical analyses were performed using one-way ANOVA ; however, the iPCR assay was able to measure CXCL13 concentrations in 81% (43 out of 53) of all CSF samples tested as compared to only 29% (11 out of 38) with the commercial ELISA  and a highly sensitive iPCR assay with excellent sensitivity and performance with CSF samples (LLOQ =1 pg/mL). For CSF samples with measurable CXCL13 concentrations by ELISA (>15.6 pg/mL), an excellent correlation was observed between the ELISA and the iPCR ELISA . Among the lymphoid chemokines, similar patterns were seen with CXCL12, CXCL13, CCL19, while CCL21 was not coupled. When individual inflammatory and lymphoid chemokines were compared, a correlation was observed between CCL19 and CXCL10 or CXCL9   and was poorer with QIgA or QIgG  and poorly with QIgA (r = 0.27) (data not shown). When each cohort was examined separately, CCL19 was decreased in SPMS despite retention of elevated QIgG values  and 9 RRMS subjects undergoing an exacerbation had a lumbar puncture and then received treatment with high dose IVMP. A second lumbar puncture was performed one month after treatment. In general, the chemokine levels were stable over time . In seveThe goal of this study was to explore measurement and analysis methods to compare the levels of chemokines in both the serum and CSF in the relapsing and progressive phases of MS. Specifically, we wished to develop more robust metrics that could serve as surrogate indicators for meningeal/cortical inflammation in SPMS in the context of clinical trials. The composite chemokine scores defined here are promising and can be applied to future studies. In these small cohorts of patients with relatively advanced disease, neither group of chemokines tracked preferentially with one particular disease stage. Heterogeneity in disease status, medication usage, advanced disease or substantial contributions of subclinical disease may have masked potential differences in the chemokine patterns and larger studies would be required to elucidate this important question. This study confirms and extends previous observations demonstrating that both inflammatory and lymphoid chemokines are elevated in the CSF in RRMS especially during exacerbation ,39,41,42Barring the small size of this feasibility study, there are several scenarios that could explain the lack of qualitative differences in the CSF chemokine signatures between the stable RRMS and SPMS subjects we studied. First, the general notion that lymphoid chemokines are preferentially associated with meningeal inflammation as opposed to CNS inflammation in general in MS could be erroneous. Second, the contribution of the progressive cortical component of the disease in SPMS may be small relative to the classical white matter component. Third, the nature of the immunology/inflammation could be very similar at these two stages at least as reflected in the CSF chemokine milieu. Given the presence of substantial cortical inflammation early in the disease process i.e. at the CIS stage, multiple pathologies may already be present during the relapsing phase of the disease making the differentiation of RRMS from SPMS by CSF chemokine signatures not possible . There were some surprises in this analysis. CCL19 and CCL21 are in theory both lymphoid, yet tracked differently in this study. Both chemokines interact similarly with CCR7 to promote naïve and central memory T cell trafficking. CCL21 is efficiently immobilized onto matrix and in this state can drive T cell crawling along reticular networks or lymphocyte entry into lymphatic compartments. In contrast CCL19 remains more soluble and therefore may report more accurately on relative levels of immune activity. The apparent increased consumption of CCL21 in the face of increased CSF CCL19 could be consistent with these differing biochemical properties. CXCL12 is produced by the vasculature and astrocytes and is key for retention of lymphocytes within the perivascular compartment . Loss ofIgM values, an observation that could be consistent with the recent suggestion that IgM oligoclonal bands may track with brain atrophy and lesion load [CXCL13 levels in CSF have been widely explored and they have the potential to reflect B cell involvement in MS. Indeed, both total cellularity and B cell content in the CSF correlated well with CXCL13 concentrations ,23. In tion load ,49. We fThe CSF to serum ratios in the normal subjects for these chemokines, BAFF, albumin, IgG, IgA and IgM were roughly consistent with a simple size dependence for the blood-CSF barrier. However, CCL2 and CXCL10 (but not CXCL9) as well as BAFF had higher CSF/serum ratios when compared to the other chemokines, suggesting intrathecal production in the normal CNS. The production of substantial amounts of BAFF in the normal CNS has been well documented . The CXCIn summary, we developed robust methods for the measurement and analysis of inflammatory and lymphoid chemokines in the CSF of MS patients. Results of a pilot study in a small number of subjects from a single clinic failed to reveal major differences between stable RRMS and SPMS. Composite “chemokine scores” may form the basis for more usable clinical biomarkers and patient stratification in stable RRMS and SPMS subjects and warrant further evaluation in larger cohorts of patients and possibly in clinical trials. File S1Figures S1-S5.(PDF)Click here for additional data file.


Cytokines exhibit a pleiotropic effect in the regulation of the immune cell function, tumor growth and antitumor immune responses. A total of 30 patients with colorectal carcinoma were enrolled on this study and their levels of interleukin (IL)-1β, IL-6, IL-8, tumor necrosis factor (TNF)-α, serum granulocyte colony-stimulating factor (sG-CSF) and serum macrophage colony-stimulating factor (sM-CSF) were measured preoperatively using ELISA. Tumor-infiltrating granulocyte (TIG), tumor-associated macrophage (TAM), G-CSF and M-CSF expression in tumor cells were examined using immunostaining. This study revealed abnormal levels of cytokines in patients, including IL-1β , IL-6 (16/30 53.3%), IL-81 , TNF-α , sG-CSF  and sM-CSF . There was a positive linear correlation between IL-6 and sM-CSF . sG-CSF was significantly associated with a deeper tumor invasion (P=0.039) and a more advanced tumor stage (P=0.023). The granulocyte/lymphocyte (G/L) ratio was associated with abnormal levels of sG-CSF. Logistic univariate analysis revealed that TIGs were a risk factor for lymph node metastasis (0.019) and TAMs were a risk factor for depth of invasion (0.029), but this was not confirmed in logistic multivariate analysis. In conclusion, IL-6, IL-8, sM-CSF and sG-CSF may indirectly promote tumor growth, progression and metastasis by changing the leukocyte populations in the blood and the tumor microenvironment. Cytokines exhibit pleiotropic effects and are crucial for the regulation of cell growth and differentiation. Previous studies have evaluated the cytokine network, which is involved in the local inflammatory and immune responses against tumors –4. CytokTumor cells are known to produce various cytokines and chemokines that attract leukocytes and promote their transformation into one of the diverse leukocyte populations, mainly granulocytes, monocytes/macrophages, dendritic cells (DCs) and lymphocytes. Subsequently, each population is able to produce an array of cytokines to allow it to escape the immune control of the host ,10. CytoIn the present study, we measured the level of pro-inflammatory cytokines, tumor infiltrating granulocytes (TIGs) and tumor-associated macrophages (TAMs), determined the granulocyte/lymphocyte (G/L) ratio and examined the expression of granulocyte colony-stimulating factor (tG-CSF) and macrophage colony-stimulating factor (tM-CSF) in tumor cells to evaluate their role in tumor progression.A total of 30 patients with primary colorectal carcinoma, who underwent curative surgical treatment between January 2008 and December 2011 at the Department of Surgery, First Affiliated Hospital of Dalian Medical University, Dalian, China, were included in the study. All patients received fasting hemospasia to determine the quantity of cytokines preoperatively. The tumors were histopathologically classified according to the 1997 tumor node metastasis (TNM) classification, as recommended by the International Union Against Cancer (5th edition). None of the patients had received chemotherapy or radiotherapy prior to surgery.The levels of immunoreactive serum G-CSF (sG-CSF), sM-CSF, interleukin (IL)-1β, IL-6, IL-8 and tumor necrosis factor (TNF)-α were measured using ELISA. The following commercially available ELISA kits were used: IL-1β, IL-6, TNF-α, M-CSF and G-CSF  and IL-8 . The procedures for the cytokine assays were carried out according to the manufacturer's instructions.μm) containing the tumor margin were applied. TIGs and TAMs were detected with mouse anti-granulocyte  and monoclonal mouse anti-CD68  antibodies. Negative control sections were stained by omitting the primary antibody.Immunostaining procedures for TIGs and TAMs were performed using the EnVision™+/HRP method  with heat-induced antigen retrieval. Paraffin sections  and monoclonal anti-M-CSF  antibodies. Negative control sections were stained by omitting the primary antibody. Specimens were considered to be positive for tG-CSF and tM-CSF when ≥20% of the tumor cells exhibited positive immunoreactivity.All data, including stage of disease and pathological factors, were obtained from clinical and pathological records. Pearson's correlation coefficient and Spearman's correlation were used to assess the correlation between related variables. Significant differences were determined using the Kruskal-Wallis test. Binary logistic regression analysis was employed to determine the significant predictors for lymph node metastasis and depth of tumor invasion. P<0.05 was considered to indicate a statistically significant difference.A total of 30 patients underwent preoperative blood sampling, however the data for sM-CSF and TNF-α were lost for 9 patients. Abnormal levels  of IL-1β, IL-6, IL-8, TNF-α, sG-CSF and sM-CSF were detected in 1/30 (3.3%), 16/30 (53.3%), 15/30 (50%), 4/21(19%), 17/30 (56.7%) and 4/21 (19%) cases, respectively.The average number of TIGs and TAMs was 47.3±26.1 and 32.5±33.8 per 10×40 hpf, respectively. TIGs were mainly located at the site of tumor invasion, however TAMs were located in areas of tumor invasion and the stroma . GranuloWe analyzed the correlation between all the cytokines which feature in this study. Pearson's correlation coefficient test revealed that there was a positive linear correlation between IL-6 and sM-CSF  and Spearman's correlation test revealed that there was a significant correlation between IL-6 and sG-CSF . However, no significant correlation was identified between the remaining combinations of cytokines .We investigated the association between the tumor stage/cytokines and the G/L ratio; a significant correlation was identified for tumor stage , the G/L ratio was significantly increased in the abnormal sG-CSF level group (P=0.049), the level of IL-8 was significantly decreased in the abnormal sG-CSF level group (P=0.026) and TAM levels were significantly increased in the abnormal IL-8 level group P=0.037; .Analysis revealed that sG-CSF was significantly associated with the depth of tumor invasion (P=0.039) and a more advanced tumor stage (P=0.023). However, significant correlation was not identified between cytokines and lymph node metastasis and between TIGs or TAMs and tumor stage, depth of invasion and lymph node metastasis .Immunostaining, which used antibodies against tM-CSF and tG-CSF , revealeLogistic univariate analysis revealed that TIGs were correlated with lymph node metastasis, however, in logistic multivariate analysis, they were not identified as a significant independent risk factor for lymph node metastasis (P=0.069). Logistic univariate analysis also revealed that levels of TAMs were correlated with depth of invasion, however, in logistic multivariate analysis, they were not identified as an independent risk factor for depth of invasion P=0.063; .This study aimed to clarify the correlation between pro-inflammatory cytokines, immune cells and tumors. This has previously been investigated by numerous studies, which showed that cytokines regulate cell growth and, more importantly, cell proliferation, and they may be produced by tumor or immune cells. However, the role of cytokines in inducing granulocytes or macrophages to become involved in the anti-tumor immune response was unclear.et al cases, however, only a few cases of G-CSF-producing colorectal carcinomas have ever been reported. We also examined tG-CSF expression in tumor cells using immunohistochemical staining. A reaction for tG-CSF was observed in only 5 cases and this was not associated with the level of sG-CSF (P=0.498). The levels of immunoreactive sM-CSF, IL-1β, IL-6, IL-8 and TNF-α were measured by ELISA and a positive linear correlation was identified between the levels of IL-6 and sM-CSF. The level of sG-CSF was significantly increased in the abnormal IL-6 level group. M-CSF expression in tumor cells was examined using immunostaining and immunoreactivity was observed in 17/30 (56.7%) cases, which is similar to results from previous studies on M-CSF production by tumor cells –16. Howeet al. Sato et al(et al demonstr al(et al–24.Therefore, we measured the G/L ratio, which represents the relative number of these two major leukocyte populations, indicates fluctuations in their numbers and reveals their potential impact on the progression and prognosis of cancer. Hence, the G/L ratio, which is easily measured in a clinical setting, is a valuable indicator of tumor progression  and is uIn carcinogenesis, cytokines and chemokines from tumor cells are able to build a supportive microenvironment and induce inflammatory cells, which contribute to tumor growth, progression and metastasis –29. In tDue to the pleiotropy of cytokines, it is difficult to confirm the roles of cytokines in the growth and progression of tumors. However, the present study reveals a correlation between tumors and immune cells, which secrete cytokines, and this suggests that cytokines may indirectly promote the growth, progression and metastasis of tumors..


The central nervous system can act as a compartment in which HIV can replicate independently from plasma, and also as a sanctuary in which, under suboptimal drug pressure, HIV antiretroviral genetic variants can occur. Continuous replication of HIV in brain can contribute to neurocognitive impairment. Therefore, reaching adequate concentrations of antiretrovirals in the central nervous system might be essential in providing neuroprotection and improving neurocognition. Antiretrovirals have a restricted entry into the brain, due to several factors: the unique structure of the blood-brain barrier, and the existence of efficient efflux mechanisms. However, there is a high variability of antiretrovirals in reaching therapeutic drug concentrations in cerebrospinal fluid, that depend on the characteristics of the antiretrovirals  and on their capacity to be substrate for efflux transporters. The review aims to discuss the main mechanisms that interfere with antiretroviral penetration into central nervous system, and to summarize the current data concerning the penetrability of different antiretrovirals into the cerebrospinal fluid.Abbreviations: ART = antiretroviral treatment; ARV = antiretrovirals; NRTI = nucleos(t)idic reverse-transcriptase inhibitors; NNRTI = non-nucleosidic reverse transcriptase inhibitors; INNRT = integrase inhibitors; CNS = central nervous system; BBB = blood-brain barrier; CMT = carrier-mediated transport; AET = active efflux transports; PGP = P-glycoprotein; MRP = multidrug resistance-associated proteins; SLC = solute carriers; OATP = organic anion transporting polypeptide; OAT = organic anion transporters; OCT = organic cation transporters; EFV = Efavirenz; IDV = Indinavir; ZDV = Zidovudine; d4T = Stavudine; ABC = Abacavir; ddI = Didanosine; 3TC = Lamivudine; TDF = Tenofovir; NVP = Nevirapine; PI = Protease inhibitors; APV = Amprenavir; NFV = Nelfinavir; SQV = Saquinavir; ATV = Ataznavir; TPV = Tipranavir; DRV = Darunavir; T20 = Enfuvirtide; RGV = Raltegravir  Nowadays, antiretrovirals (ARV) from six classes are available: nucleos(t)idic reverse–transcriptase inhibitors (NRTI), that interfere with reverse transcriptase by competing with the natural substrates and incorporating into viral DNA to act as chain terminators in the synthesis of proviral DNA; non–nucleosidic reverse transcriptase inhibitors (NNRTI)– that bind directly to the catalytic site of the reverse transcriptase, protease inhibitors (PI)– that inhibit the proteolitic cleavage of polipeptridic precursors, giving rise to noninfectious viral particles; integrase inhibitors (INNRT) that block the integration of proviral DNA in the cell DNA, CCR5 co–receptor antagonists that prevent interaction of the V3 loop of gp120 with the CCR5 coreceptor and fusion inhibitors that block entry of HIV into the target cell. Effectiveness of ART regimens is usually evaluated by measuring HIV RNA levels in plasma. However, there is evidence that HIV can replicate in compartments distinct from plasma [4]. Neurocognitive impairment can be the result of HIV replication in the CNS, even in patients with suppressed plasma viral loads [5] and can interfere with the patient’s functionality [6].The central nervous system (CNS) is one of the compartments in which HIV determines an autonomous infection since the early stages of infection, but it is also a sanctuary in which HIV can independently replicate and has a genetic profile distinct from plasma, due to an inadequate concentration of ARV [8]. Letendre developed and improved a quantification rank of antiretrovirals in CSF, which can be a useful tool for physicians in selecting the adequate ART for patients with neurocognitive impairment [9].There seems to be a direct correlation between the concentrations of ARV in the CSF and the decrease of HIV CSF viral load .The access of various molecules into the CNS is closely related to their ability to penetrate through the blood-brain barrier (BBB). The BBB consists of several compounds, two of which are better described: the vascular BBB primarily includes the cerebral capillaries and the endothelial cells sealed by tight junctions and the blood-CSF barrier represented mainly by the choroid plexuses, which form the interface between the blood and the ventricular cerebrospinal fluid . Low molecular weight and higher lipid solubility facilitate a better penetration through the BBB. Conversely, high protein binding rates associated with low free drug levels diminish the penetration through the BBB.Diffusion is one of the most common mechanisms of crossing the BBB [12]. CMT systems are generally responsible for the transport of nutrients  across the BBB. RMT are used to carry endogenous large–molecule neuro–peptides, mainly hormones, into the brain by specialized ligand–specific receptor systems, including the insulin receptor, the transferrin receptor, the insulin–like growth factor receptor, the leptin receptor, the neonatal Fc receptor or the type BI scavenger receptor [13]. However, the most important transporter mechanisms from the ARV perspective are the AET. Active efflux transporters limit the brain uptake of several high lipophilic drugs [14]. P–glycoprotein (PGP), multidrug resistance–associated proteins (MRP) are the best described energy–dependent efflux transporters that represent a major obstacle for ARV penetration in CNS. Solute carriers (SLC) are another group of efflux transporters especially designated for anions [15]. Members of this group include organic anion transporting polypeptide (OATP), organic anion transporters (OAT) and organic cation transporters (OCT). All these polypeptides are Na– and ATP–independent and they are expressed on the cell membrane of the endothelial capillaries in the brain. Choroid plexuses are also significantly involved in the cellular uptake of drugs, including ARV in the brain [16].The efflux or influx transport mechanisms also play a crucial role in drug penetration into the CNS. The transporters are classified as carrier–mediated transport (CMT), active efflux transports (AET), and receptor–mediated transport (RMT) . Considering the same parameters (molecular weight and protein binding), nevirapine from the NNRTI class and indinavir from the PI class have the best probability of reaching good CSF levels.Zidovudine (ZDV) has the best partition coefficient in the brain and cerebral tissue, reflecting the lipid solubility of the compound [17]. ZDV is substrate for PGP, MRP-4 and MRP-5 [18]. One of the first studies on ZDV in CSF, demonstrated that penetration of ZDV into the CSF appeared to be dose independent, which may be an explanation for the efficacy of low doses of ZDV in the prevention and treatment of HIV–related neurological diseases [19]. Since the beginning of the HIV epidemic, treatment with ZDV was associated with decreased HIV RNA loads in CSF, less alteration of the brain tissue and improvement of neurocognitive performance in children with HIV encephalopathy [20].Stavudine (d4T) CSF stavudine concentrations reached or exceeded the mean concentration, producing 50% of the maximal effect in vivo (EC 50) for HIV. Oatp–2 like transporter has been implicated in its uptake [21]. ENT1 and ENT2 have been suggested as transporters for d4T on animal models [22].Abacavir (ABC) has moderate plasma protein binding and lipid solubility which account for a good CSF penetration rank. Animal models have shown that ABC reaches the brain, but not the CSF by a non–saturable mechanism, meaning that its transport across the BBB is not influenced by other drugs [23]. In an pharmacokinetics study on paired plasma–CSF from 54 patients, Caparelli et. al demonstrated a mean CSF/plasma ratio for ABC of 36% that increased progressively with the augmentation of the doses, and showed that ABC penetrated into the CSF where it reached adequate concentrations which were able to inhibit the replication of HIV in the CNS [24]. Although a better CSF penetration compared to other antiretrovirals has been demonstrated for ABC, its lower penetration rate as compared to the corresponding free fraction indicated the possible existence of other active efflux mechanisms. In 2008, indeed, Giri et. al., demonstrated that PGP is the dominant transporter limiting the CNS penetration of ABC [25].Didanosine (ddI) was able to cross the BBB using saturable and non–saturable mechanisms [26]. ddI transport from the blood into the choroid plexuses involves an OATP 2–like transporter. In patients with HIV infection, ddI reaches relatively good CSF concentrations [27].In animal models, Lamivudine (3TC) movement across the blood–CSF barrier was examined in an isolated choroid plexus model, which showed low CSF accumulation of this molecule [28]. However, 3TC uptake from blood into the choroid plexus was significant, and it was facilitated by a digoxin–sensitive transporter. In the same study, 3TC had no major interactions with ABC. Based on rat models it is speculated that OCTs and probably OACT are involved in the cellular uptake of ZDV and 3TC [29].Tenofovir (TDF) reaches a CSF concentration of only 4% of the plasma concentration, suggesting a passive or limited active transport and probably active efflux mechanisms from the CSF. These concentrations did not exceed IC50 for the wild–type; therefore, TDF is not suited for controlling CSF HIV replication [30]. However, studies on animal models demonstrated that although the transport of TDF in the CSF is minimal, the TDF precursor, PMPA, can reach the brain and accumulate in the choroid plexuses. These observations are related to the hydrophilic nature of PMPA and indicate the possible existence of a transporter at the choroid plexus site [31].Efavirenz (EFV) in CSF is limited. The efflux mechanism described was the induction of the expression and function of PGP [32]. EFV also inhibits MRP–1, MRP–2 and MRP–3 in a concentration–dependent manner [33]. In a small study on 9 patients, EFV levels were constantly less than 1%  of plasma levels [34]. However, in the same study, the authors found a mean EFV concentration of 35.1 nM (range 6.6–58.9 nM), that was above the IC95 of HIV wild type. This indicated that even if EFV was present in the CSF at low levels, these levels were effective in suppressing CSF viral levels when used in combination therapy. In a more recent report, Best et.al [35] have demonstrated that, even if EFV concentrations in CSF were only 0.5% of plasma concentrations they exceeded IC50 for the wild–type virus and were enough to inhibit the replication of HIV in the CSF.Important differences have been observed among the NNRTI class representatives, concerning the penetration in CNS. It has been demonstrated that penetrability of Nevirapine (NVP) crosses well the BBB [36], and maintains concentrations in CSF that remain stable in time [37]. NVP had the highest CSF/plasma penetrability rate when compared to other drugs [38]. NVP has the highest penetrability rank in Letendre’s classification [9], and therefore can be used in patients with neurocognitive impairment [39].Protease inhibitors (PI) have good lipid solubility, and therefore they were expected to concentrate adequately in the brain, but, in fact, the CSF concentrations of these drugs are low [40]. The explanations for the limited penetrability of PIs in the CSF are: the presence of efflux mechanisms  and high plasma protein binding (with the exception of Indinavir). Boosting with low–dose ritonavir enhances PIs’ penetrability into the CSF [8].Indinavir (IDV) has the best CSF concentrations among all PIs [44]. This advantage can be explained by low protein–binding. Median concentration of IDV in CSF was 210 nmol/l [45], which is the threshold for IC95 in vitro. IDV is essentially the only PI that reaches CSF concentrations above IC95 [46] [42]. From a clinical point of view, the presence of IDV in CSF was associated with significant improvement of neurocognitive performances [47].Lopinavir (LPV/r) – Kaletra, one of the best characterized PIs in terms of CSF penetrability. Although LPV/r has a CSF/plasma penetration rate of 0.22, its CSF levels are above IC50, [49]. Moreover, patients with monotherapy with LPV/r, and those with cART containing LPV/r [50] have a good decrease of HIV RNA in CSF and plasma and a reduction of the immune activation [51]. Because it penetrates the central nervous system in therapeutic concentrations and appears to reduce HIV replication in the central nervous system, LPV/r may benefit subjects who receive a diagnosis of or are at risk for HIV–associated neurocognitive disorders [51]. Similarly, Amprenavir (APV) boosted with low–dose RTV reaches CSF concentrations above IC50. In a study presented by Letendre et. al. [52], boosted or unboosted FPV had concentrations higher than IC50 with a CSF/plasma ratio of 0.45 – 1.30.Concerning the other PIs, it has been demonstrated that their concentrations in CSF are enough to control viral replication although some of them concentrate poorly in CSF compared to plasma due to limited binding to plasma proteins, high molecular weight and efflux mechanisms. A good example regarding this issue, is boosted Nelfinavir (NFV), Saquinavir (SQV), Ataznavir (ATV) and Tipranavir (TPV) do not reach therapeutical concentrations in CSF [9]. CSF levels for NFV [53] and for SQV and RTV were below the detection limit [54]. For ATV, the CSF concentrations were highly variable and 100–fold lower than plasma concentrations, even with ritonavir boosting. CSF concentrations of ATV do not consistently exceed the wild–type IC50 of atazanavir and may not protect against HIV replication in the CSF [55].Some PIs like 56] found detectable CSF Darunavir (DRV) levels in all the assessed patients. Most of them exceeded or remained in the same range as levels needed to inhibit the replication of the wild type virus, making it probable that DRV, at least to some extent, contribute to the suppression of HIV replication in the central nervous system.In a recent study, Yilmaz et. al. . Cases of virological failure secondary to selection of resistance mutations to T20 in CSF with consecutive loss of viral suppression in plasma have been described [58]. Still, T20 is recommended for patients with HIV related neurocognitive impairment for its effect on the plasmatic pool, preferably in association with other three antiretrovirals with good CSF penetrability [59]. The motivation of this approach is due to the fact that antiretroviral treatment reduces immune activation with an indirect effect on the CNS in addition to the control of HIV replication in the plasma compartment [60].Concerning the new antiretroviral drugs, data available to date suggest a good penetrability of integrase inhibitor and CCR5 co–receptors in the CSF.The penetrability of Raltegravir (RGV) demonstrated a good passage into CSF. In a study published in 2009 by Yilmaz and her collaborators, 50% of the CSF samples exceeded IC95. In addition to contributing to the control of systemic HIV–1 infection, raltegravir reaches local inhibitory concentrations in CSF in most, but not all, patients [61]. In a similar study, Letendre [62] detected RGV levels above IC50 in all CSF samples of 21 patients and the median concentration of RGV in the CSF was 14.5 ng/mL . The median plasma concentrations were 260.9 ng/mL . The CSF/plasma ratio was 5.8% . Based on these results, RGV can be used as a component of regimens with good CSF penetrability.65]. Tropism discordances between viral populations present in the CSF and in plasma have been observed. R5–tropic virus was usually found in the CSF [67]. Patients with advanced HIV disease [68] and especially those with HIV associated dementia had evidence of macrophage tropism and CCR5 use [70], but strains that use both CCR5 and CXCR4 co–receptors for cell entry have been identified in the brains of some individuals [72]. However, it seems that brain–derived Env’s still had preferential CCR5 usage [72].Studies regarding HIV tropism and coreceptors usage in the brain cells are limited. As microglia and monocyte–derived macrophages are the support of productive HIV infection in the CNS, and most likely play a major role in the development of HIV dementia, several studies were focused on the coreceptors used by these cells and they found that CCR5 is the main coreceptor used by HIV–1 isolated from the brain [75]. Based on the observations that CCR5 receptors are mostly used in the brain of HIV–infected individuals and on studies that demonstrate that Maraviroc can reach adequate concentrations in CSF (above IC50) [77], the use of Maraviroc might be beneficial in patients with HIV–associated CNS diseases, especially in those with neurocognitive impairment. Nevertheless, clinical trials are needed to demonstrate the effectiveness of Maraviroc in the brain and in order to answer the question if the brain can be considered a potential reservoir of CXCR4 after exposure to CCR5 inhibitors.The clinical consequences of virus populations with different tropism in specific body and cellular compartments as compared to the viral population in plasma are poorly understood [78] or too expensive (i.e. nanotechnology) [80]. On the other hand adjunctive therapies, like valoproic acid, lithium [81], minocycline [84] and other small molecules showed encouraging results in terms of improving neurocognition but are lacking longitudinal follow–up studies [85]. For example, minocycline, a second–generation tetracycline derivative, that has increased penetration in CNS, has been shown to decrease both the virus load in the cerebrospinal fluid and the brain, as well as the severity of CNS disease in a simian immunodeficiency virus macaque model of HIV–associated neurological disease [82]. These, together with its immunomodulatory properties that reduce chronic activation and inflammatory processes may make it a promising adjuvant in active antiretroviral therapy.As antiretroviral drugs have limited penetration into the CSF, other alternative mechanisms have been studied in order to limit the viral replication and to provide neuroprotection. The alternatives designed “to cheat” the diffusion or efflux mechanisms are either ineffective [The efficacy of antiretroviral regimens in the CNS should concern every physician involved in the care of HIV–infected patients. This has become a requirement, as HIV–associated neurocognitive impairment nowadays involves mostly mild and moderate forms. Therefore, the diagnosis of neurocognitive impairment in an early stage is needed in order to give an ART regimen with good CSF penetrability rank, which will presumptively assure a degree of neuroprotection.Compartmentalization of HIV infection with genetic differences between plasma and CSF strains requires the quantification of HIV RNA and HIV resistance profile in both plasma and CSF and adjustment of ART regimen according to both results.The evaluation of efficacy of an antiretroviral regimen in CNS sanctuary requires beyond awareness on the penetrability and resistance mutations of ART in CSF, considering other factors as modification of BBB (e.g. inflammation), drug interactions and co–morbidities. CNS is a compartment where HIV enters in the early stages of infection and can undergo neuro–adaptation. The unique structure of BBB and the existence of well–organized efflux mechanisms block or severely limit the access of ART. Approaching this sanctuary is possible by medication with good lipid solubility, low molecular weight, and low plasma protein–binding that cannot be accessed by efflux transporters (either by avoiding or by blocking them). However, further studies are needed to find an ideal drug to fulfil these criteria and assure a good balance between therapeutic effect and neurotoxicity.Longitudinal studies are needed to clarify if a neuro–cART can be protective for neurocognitive impairment in adults but also in the developing brain of children.