Plasmodium falciparum histidine rich protein-2 (PfHRP-2) and clinical, laboratory and radiographic features in a cohort of children with retinopathy-positive CM.Cerebral malaria (CM) causes a rapidly developing coma, and remains a major contributor to morbidity and mortality in malaria-endemic regions. This study sought to determine the relationship between cerebrospinal fluid (CSF) Patients included in the study were admitted (2009–2013) to the Pediatric Research Ward  meeting World Health Organization criteria for CM with findings of malarial retinopathy. Enzyme-linked immunosorbent assay was used to determine plasma and CSF PfHRP-2 levels. Wilcoxon rank-sum tests and multivariable logistic regression analysis assessed the association of clinical and radiographic characteristics with the primary outcome of death during hospitalization.In this cohort of 94 patients, median age was 44 (interquartile range 29–62) months, 53 (56.4%) patients were male, 6 (7%) were HIV-infected, and 10 (11%) died during hospitalization. Elevated concentrations of plasma lactate (p = 0.005) and CSF PfHRP-2 (p = 0.04) were significantly associated with death. On multivariable analysis, higher PfHRP-2 in the CSF was associated with death  while plasma PfHRP-2 was not .Elevation of CSF, but not plasma PfHRP-2, is associated with death in this paediatric CM cohort. PfHRP-2 egress into the CSF may represent alteration of blood brain barrier permeability related to the sequestration of parasitized erythrocytes in the cerebral microvasculature.The online version of this article (10.1186/s12936-018-2272-y) contains supplementary material, which is available to authorized users. Plasmodium falciparum parasitaemia and no other discernible cause of coma  were excluded from the study to avoid plasma pfHRP-2 contamination of CSF. All children received standard of clinical care while on the inpatient ward, including appropriate anti-malarial, antipyretic and anticonvulsant medications.Demographic information was collected and human immunodeficiency (HIV) testing performed on admission on all patients whose guardians gave consent. Cases were identified as malarial retinopathy-positive based on at least one of the following changes in the optic fundus on indirect ophthalmoscopy examination, performed by an ophthalmologist with expertise in the diagnosis of malarial retinopathy: retinal haemorrhages, retinal whitening, and/or orange or white vessel discoloration. Patients with evidence of papilloedema on admission were excluded from the study as they did not undergo LP. Venous blood was drawn on admission for subsequent PfHRP-2 analysis, and finger-prick samples were analysed to determine parasite species and density, packed cell volume, blood glucose, and lactate concentrations. Additional laboratory parameters analysed on admission included white blood cell (WBC) count, platelet count, quantitative peripheral parasitaemia, CSF WBC count, and CSF protein. A sub-set of patients underwent brain MRI during their hospital admission. Ninety per cent of scans were performed within the first 24 h of hospitalization. Scanning was performed with a General Electric Signa Ovation Excite 0.35T Magnet . Details of the scanning protocols used for the patients are found elsewhere . Two radThe primary clinical outcome of interest was death during hospitalization. Co-variables of interest included known laboratory markers of poor outcome in severe malaria including age, admission lactate, WBC count, and severely increased brain volume score on MRI.Venous blood was drawn and CSF obtained at the time of admission. CSF and plasma were both stored at − 80 °C until the time of analysis. Aliquots of CSF or plasma were thawed and analysed in batches. Plasma or CSF were diluted at a ratio of either 1:100 or 1:500 in phosphate-buffered saline and tested as previously described .Patient demographic and clinical characteristics were summarized using appropriate descriptive statistics, e.g., counts and percentages, means and standard deviations (SDs), and medians and interquartile ranges (IQRs). Non-parametric analysis methods were used due to non-Gaussian distribution of data. Characteristics of patients who survived to discharge and those who died during hospitalization were compared using Fisher’s exact tests or Wilcoxon rank-sum tests. Wilcoxon signed-rank tests were used to determine the statistical differences between levels of PfHRP-2 in paired CSF and plasma samples. Spearman correlation was used to determine the relationship between CSF PfHRP-2 levels and MRI features of interest. Sensitivity, specificity and exact binomial 95% confidence intervals (CIs) for each variable of interest were calculated. Univariable logistic regression analysis examined clinical and radiographic characteristics with relationship to the level of PfHRP-2 in plasma, CSF or the CSF/plasma ratio. Multivariable logistic regression was performed to examine the association between potential predictors and the likelihood of death. Variables included in the multivariable logistic analysis were those determined a priori based on clinical relevance; age (6 month increments), abnormal WBC count on admission , and abnormal admission lactate (lactate > 5 mmol/L). Odds ratios (ORs) and 95% CIs were used to quantify the strength of these associations. Analyses were performed using SAS version 9.3 . All tests were two-sided and considered significant at p < 0.05.3. Laboratory parameters stratified by outcome are shown in Table There were 450 CM admissions to the malaria research ward during the period of 2009–2013, 349 (77.6%) of which had evidence of malarial retinopathy. Forty-nine patients were excluded due to traumatic LPs. LP was not performed in 80 individuals, due to papilloedema on ophthalmoscopy assessment or if the child was deemed too clinically unstable to undergo the procedure. In 126 patients, CSF samples were not available from patients who had an LP performed prior to admission to the malaria research ward (either on the paediatric inpatient ward or in the emergency department). Paired plasma and CSF samples from non-traumatic LPs were available in 94 retinopathy-positive CM patients  patients had brain MRIs performed. On univariable analysis, severely increased brain volume, defined by brain volume scores of 7 or 8, was associated with death (p = 0.048). Brain volume scores of 1–6 were significantly associated with higher CSF PfHRP-2 compared to scores of 7–8 (p = 0.01)  and CSF PfHRP-2 p = 0.04) were significantly associated with death while other clinical and-laboratory parameters were not associated with mortality Fig. , Table 2 were sigIn this study, assessment of the levels of PfHRP-2 was performed in the CSF and plasma in retinopathy-positive children with paediatric CM to determine whether PfHRP-2 is associated with clinical, laboratory and radiographic features during acute illness. Peripheral blood parasitaemia and plasma PfHRP-2 have often been used as a marker for disease severity, although not associated with mortality in this study –22, 24. P. falciparum, the most pathogenic of the human Plasmodium species, has proposed functions which include binding zinc and haem, mediating formation of haemozoin as well as immunomodulatory effects and perturbations of the coagulation cascade [The biologic significance of increased PfHRP-2 in the CSF remains unclear. Many factors likely contribute to CM pathogenesis; the mechanisms that lead to death are incompletely understood. PfHRP-2, which is unique to  cascade –35. Rece cascade . FurtherIn this study, comparison of various clinical and radiographic features in relation to plasma and CSF PfHRP-2 and mortality was also performed. A previous study in Nigerian children with CM found that age under 3 years, abnormal breathing pattern, hypoglycaemia and leukocytosis were predictive of mortality . In a laThere are several strengths of the study. PfHRP-2 was tested in a large cohort of paediatric CM patients, with extensive clinical, laboratory and radiographic characterization. Examination of retinopathy-positive CM cases, which represent a cohort of true CM cases, with eye examinations documented by trained ophthalmologists was performed. Paired plasma and CSF samples allowed for direct comparisons of plasma and CSF pfHRP-2. Additionally, detailed clinical, laboratory and radiographic interpretation was available in a large group of paediatric patients, which has not been previously described with relation to CSF or plasma biomarkers.There were several limitations to the study. The sample excluded subjects with papilloedema, as they did not undergo LP. However, even after excluding these cases, a relationship between CSF PfHRP-2 and outcome was identified. Also, many cases were excluded because children had an LP prior to admission to the malaria research ward, resulting in the CSF sample being unavailable for PfHRP-2 analysis. A selected group who had MRI scans was examined and results related to neuroimaging findings may be influenced by the selected patients, who were able to undergo neuroimaging. There was no evaluation of PfHRP-2 with respect to longitudinal neurological outcome as these data were not available in the patient cohort, though this would be valuable to investigate as children with CM often have neurological sequelae. There was no evaluation of plasma and CSF PfHRP-2 in parasitaemic children with no neurological symptoms, as doing an LP in that setting would be unethical. Based on previous observations though, it may be important to quantify CSF PfHRP-2 in those children with less severe malaria infection and those with severe, non-cerebral malaria, including those who are comatose though do not have findings of malarial retinopathy. Additionally, the focus here was on CSF PfHRP-2 but examining other markers of BCSFB and BBB function would be insightful.The study results demonstrate that there is a significant relationship between CSF PfHRP-2 and mortality in paediatric cases of retinopathy-positive CM. Previous studies have found that plasma PfHRP-2 is associated with mortality in patients with severe malaria , 24. ThiAdditional file 1: Table S1. Clinical and laboratory characteristics of patients included in and excluded from the study.


It was our purpose to identify vulnerable plaques in the thoracic aorta using 3D multi-contrast CMR and estimate the risk of cerebral embolization using 4D flow CMR in cryptogenic stroke patients and controls.One hundred patients  underwent a novel 3D multi-contrast  CMR protocol at 3 Tesla for plaque detection and characterization within the thoracic aorta, which was combined with 4D flow CMR for mapping potential embolization pathways. Plaque morphology was assessed in consensus reading by two investigators and classified according to the modified American-Heart-Association (AHA) classification of atherosclerotic plaques.p = 0.81]. However, plaques ≥4 mm were more frequent in stroke patients . Of those patients with plaques ≥4 mm, seven (17.5%) stroke patients and two (3.3%) controls (p < 0.001) had potentially vulnerable AHA type VI plaques. Six stroke patients with vulnerable AHA type VI plaques ≥4 mm had potential embolization pathways connecting the plaque, located in the aortic arch (n = 3) and proximal descending aorta (n = 3), with the individual territory of stroke, which made them the most likely source of stroke in those patients.In the thoracic aorta, plaques <4 mm thickness were found in a similar number of stroke patients and controls [23 (57.5%) versus 33 (55.0%); Our findings underline the significance of ≥4 mm thick and vulnerable plaques in the aortic arch and descending aorta as a relevant etiology of stroke.DRKS00006234; date of registration: 11/06/2014Unique identifier: The online version of this article (doi:10.1186/s12968-017-0379-x) contains supplementary material, which is available to authorized users. Atherosclerosis of the aorta is regarded as a source of embolic stroke, if complex plaques  are detected in the ascending aorta and the aortic arch by transesophageal echocardiography (TEE) . In clinInclusion criteria for stroke patients were: a) age ≥ 50 years, b) acute brain ischemia with visible acute lesion on brain MRI, and c) cryptogenic stroke etiology after routine diagnostics including TEE. Between September 2014 and March 2015, 745 patients ≥50 years of age were admitted to our hospital for symptoms consistent with ischemic stroke and were screened for eligibility for this study within 24–48 h after admission. All patients underwent routine diagnostics .One hundred sixty nine patients had no visible brain lesion on diffusion weighted MRI and were thus excluded from the study, leaving 576 patients to be further evaluated. Of those, the stroke etiology following modified TOAST criteria was determined in 406 patients (70.5%): large-artery atherosclerosis in 195 (33.8%), cardioembolism in 141 (24.5%), small-vessel disease in 38 (6.6%) and other causes in 32 (5.6%) patients. In 170 (29.5%) patients stroke etiology remained cryptogenic despite complete and thorough diagnostic workup. We included only cryptogenic stroke patients in order to systematically evaluate the prevalence of plaques of the aortic arch and descending aorta and of potential embolization pathways that constitute a likely source of cerebral embolism. Of these 170 patients, 19 had CMR-contraindications, 23 were not legally competent to give consent due to severe stroke/aphasia, 50 required intensive care, 17 declined study participation, 10 were already transferred to another hospital or discharged before study CMR could be conducted, 8 were severely obese (BMI ≥35) and thus not suitable for our CMR protocol. Accordingly, 43 patients were scheduled for a study CMR. One patient aborted the examination and two had to be excluded because atrial fibrillation was detected after performance of study CMR (=determined stroke etiology). Thus, complete CMR data of 40 acute stroke patients were available for final analysis.Cardiovascular risk factors and National Institutes of Health Stroke Scale (NIHSS) were prospectively documented in all participants based on personal interviews and patients’ medical charts.n = 60 in the control group with a power of 80%, alpha error of 0.05, and an enrollment ratio of 1.5. Accordingly, 60 controls were frequency matched to stroke patients in terms of age, sex, and hypertension and underwent CMR. Cardiovascular risk factors were prospectively documented in all participants based on personal interviews and patients’ medical charts.Patients from the Eye Center without a history of retinal or cerebral ischemia as indicated by personal interview and patients’ medical charts were chosen as controls. Based on data from a previous study  [The study was approved by the University of Freiburg Ethics Committee and informed consent was obtained from all participants.All CMR examinations were executed on a 3 Tesla system  using a 12-element body coil. All sequences were ECG-synchronized and respiration-controlled using navigator-gating  and triggering , respectively.R = 2, 32 ref. lines), a T2w black-blood , and a PDw black-blood  sequence. All multi-contrast sequences had the same geometric parameters: Field-of-view (FOV) = 264 × 240 mm2, matrix = 256 × 178, 56 partitions resulting in a nearly-isotropic spatial resolution of 1.15 × 1.15 × 1.17 mm3. T1w bright-blood images of the aorta were acquired using a RF spoiled GRE sequence with 41 segments per cardiac cycle. For fat suppression, a binomial 1–2-1 water excitation pulse was used. T2w and PDw dark-blood images of the aorta were acquired using a 3D RARE sequence with variable non-selective refocusing flip angles  [The multi-contrast CMR protocol consisted of a T1w bright-blood  = 1.62 ms/6.1 ms, flip-angle = 12°, acceleration = GRAPPA (lthcare) . Efficie3, velocity sensitivity along all three directions = 150 cm/s, and parallel imaging (PEAK-GRAPPA) along the phase encoding direction (y) with an acceleration factor of R = 5 (20 reference lines).Furthermore, 4D flow CMR was used to visualize 3D blood flow within the thoracic aorta. Parameters were: TE/TR = 2.6 ms/5.1 ms, flip angle = 7°, temporal resolution = 40 ms, matrix = 320x240x58, bandwidth = 450 Hz/pixel, spatial resolution =2.1 × 2.1 × 2.5mmAll datasets were pseudonymized and analyzed in consensus reading by two investigators with experience in CMR-based plaque analysis of five and 8 years, who were blinded to patient data and affiliation to the case or control group. In the case of a disagreement among observers, a third investigator with experience in CMR-based plaque analysis of 10 years made the decision. To analyze intra- and inter-rater variability and to avoid recognition of datasets, one observer repeated the analysis 3 months and 6 months after baseline and was blinded to results of the previous analysis. Plaques within the thoracic aorta were detected based on 3D multi-contrast CMR. The aorta was subdivided into the ascending aorta ), the aortic arch  comprising all parts of the vessel wall at equal level as the branching sites), and the descending aorta . Three-dimensional T1w, T2w, and PDw CMR images were displayed using multiplanar reformatting and synchronization to allow immediate assessment of all contrasts at one time. The DAo was scrutinized for plaques as far as 6 cm distal to the LSA.Image quality was rated separately for each sequence as good , satisfactory , and bad .Plaque thickness (i.e. distance between lumen-intima and media-adventitia interface) was quantified by manual measurements of axial reconstructions of T2w 3D images of the aorta using electronic calipers at the site of the plaques largest diameter in cross-section. Plaque composition was evaluated regarding features determining plaque vulnerability  and classified according to the modified AHA classification for CMR . The processing workflow consisted of correction of velocity offset errors and velocity aliasing artifacts, vessel segmentation and particle tracking within time-resolved and three-dimensional flow data . Two-diData are presented as mean (±standard deviations) or median (interquartile range) for continuous, absolute and relative frequencies for categorical variables. Departures from normality were detected with the Shapiro-Wilk statistic. Differences between patient groups were evaluated using chi-square tests and Fisher’s exact tests for qualitative and independent samples, t-tests for quantitative parameters. A Mood’s median test was used to test if median plaque prevalence was identical in cases and controls. The weighted Cohen’s kappa (κ) coefficient was used to quantify inter- and intra-rater agreement as excellent (κ ≥ 0.75), fair to good (κ0.40 to 0.75), and poor (κ < 0.40). All tests were two-sided with 0.05 as the level of statistical significance. Statistical analyses were performed using IBM-SPSS Statistics version 19.0.1.Demographics and cardiovascular risk factors of all study participants are provided in Table p < 0.001, 95% CI 0.67, 0.98) and intra-rater variability was ĸ = 0.96 .Duration of the complete protocol after placing the patient inside the scanner was 59:25 ± 9:27 min . Image quality was rated good in 97% and satisfactory in 3% for T1w, good in 93% and satisfactory in 7% for T2w, and good in 80% and satisfactory in 20% for PDw images. Accordingly, all sequences were assessable. Results of intra- and inter-rater agreement of AHA plaque classification were as follows: inter-rater reliability was ĸ = 0.83 (p = 0.81) had plaques of any degree (<4 mm and ≥4 mm) in the thoracic aorta. Every subject (i.e. 23 stroke patients and 33 controls) with aortic plaques had at least one small plaque (<4 mm). The median number of plaques <4 mm was 2 (range: 1–5) in stroke patients and 1 (range: 1–3) in controls (p = 0.02). Large plaques (≥4 mm) were found in 22 (55.0%) stroke patients and 10 (16.7%) controls (p < 0.001). The median number of plaques ≥4 mm was 1 (range: 1–4) in stroke patients and 1 (range: 1–2) in controls (p = 0.78).Twenty-three (57.5%) stroke patients and 33 (55.0%) controls , whilst the frequency of patients with AHA type III plaques was similar (10/40 (33.3%) vs. 26/60 (43.0%); p = 0.903). Large plaques (≥4 mm) were potentially vulnerable AHA Type VI plaques in 7/22 (31.8%) and 2/10 (20%) cases (p = 0.68).Irrespective of plaque thickness, we classified 53 and 53 plaques as AHA Type III, 8 and 0 as AHA Type IV-V, 7 and 2 as AHA Type VI (=vulnerable plaques), 12 and 6 as AHA Type VII and 5 and 1 as AHA Type VIII. Accordingly, there were more patients with at least one advanced plaque (AHA type IV-VIII) in the group of stroke patients (20/40 (50.0%)) than in controls (9/60 (15%)) (n = 10), the left (n = 4) and right (n = 3) hemisphere, and involvement of multiple vascular territories (n = 1). Of these, one patient had simultaneous plaques in the AAo, the AA, and the DAo and six had simultaneous plaques in the AA and DAo, which had a connection to the affected stroke territory.Embolization pathways connected the site of large plaques ≥4 mm with a supraaortic artery in 20 cases and 3 controls: the BCT in 12 and 2, the LCCA in 15 and 2, and the LSA in 28 and 3 , the DAo (n = 3), and in both the aortic arch and the DAo (n = 2). In the absence of a known stroke etiology, those plaques might be defined as culprit plaques, i.e. vulnerable plaques which probably caused stroke. In control patients, we detected only one potential vulnerable plaque in the aortic arch (=type VI) with a possible connection to all brain territories, and two plaques of uncertain vulnerability in the DAo (AHA type VII) with a potential retrograde embolization pathway reaching the LSA and the BCT , representative of the population at risk of the outcome, and independent of the exposure of interest. Furthermore, to our knowledge, this is the first study to apply a complete 3D multi-contrast protocol tailored to aortic plaque detection to a large group of patients. The CMR sequences used in this study showed high intra- and inter-observer agreement regarding image quality grading of the 3D T1w, T2w, and PDw sequences  and regaNo difference was observed between groups regarding the prevalence of small plaques (<4 mm thickness) throughout the aorta, which were found in more than half of all studied patients. However, despite equal frequencies of systemic cardiovascular risk factors, stroke patients had significantly more plaques ≥4 mm thickness in the entire thoracic aorta than controls, which is in accordance to the literature , but alsWithin the last years, there has been a controversy regarding the role of plaques in the DAo as a source of cerebral embolism , 16. HowIn this study, we used a novel 3-dimensional multi-contrast plaque imaging protocol to identify vulnerable plaques and 4D flow CMR to visualize potential embolization pathways from such plaques in the DAo to the brain. The multi-contrast plaque protocol comprised a T1w bright blood sequence which proved to be reliable for the detection of aortic plaques  supplemeMulti-contrast plaque CMR has already been used to classify plaques according to the AHA classification in the carotid arteries. However, AHA originally is a histological classification which was extended to CMR for carotid plaque classification after direct comparison with histologic specimen . Only onBy adding 4D flow measurements to our protocol we were able to detect potential embolization pathways to the brain. This is especially important for plaques located distal to the outlets of the brain supplying arteries in the proximal descending aorta. However, aortic flow reversal varies between individuals  and evenIdeally, a T1w dark blood sequence would have been implemented in order to better evaluate the vessel wall to further facilitate the discrimination of vessel lumen and wall. Implementation of a 3D T1w dark-blood sequence for the aorta was technically difficult to realize at that time. However, this limitation was compensated by the two additional black-blood contrasts applied during 3D T2 and PD weighted imaging in all patients. Using these three complementary CMR contrasts, we believe that even hyperintense plaque components in T1 weighted CMR were reliably discriminated from the bright blood signal. Multi-contrast plaque imaging protocols in the carotid arteries usually also comprise a time-of-flight (TOF) sequence which is used to detect fresh intra-plaque hemorrhage . HoweverIn conclusion, we have shown that our novel multi-contrast CMR protocol is capable to depict plaques in 3D and throughout the entire thoracic aorta. This protocol proved to be robust in 100 patients and should be considered for future trials in order to characterize plaques in analogy to the carotid arteries and identifying vulnerable lesions. In addition, we found that despite matching for age and cardiovascular risk factors, cryptogenic stroke patients more often had complex and vulnerable plaques in the aortic arch and the descending aorta compared to controls and thus a higher frequency of potential embolization pathways.


Donor-specific blood transfusion (DST) can lead to significant prolongation of allograft survival in experimental animal models and sometimes human recipients of solid organs. The mechanisms responsible for the beneficial effect on graft survival have been a topic of research and debate for decades and are not yet fully elucidated. Once we discover how the details of the mechanisms involved are linked, we could be within reach of a procedure making it possible to establish donor-specific tolerance with minimal or no immunosuppressive medication. Today, it is well established that CD4+Foxp3+ regulatory T cells (Tregs) are indispensable for maintaining immunological self-tolerance. A large number of animal studies have also shown that Tregs are essential for establishing and maintaining transplantation tolerance. In this paper, we present a hypothesis of one H2-haplotype-matched DST-induced transplantation tolerance (in mice). The formulated hypothesis is based on a re-interpretation of data from an immunogenetic experiment published by Niimi and colleagues in 2000. It is of importance that the naïve recipient mice in this study were never immunosuppressed and were therefore fully immune competent during the course of tolerance induction. Based on the immunological status of the recipients, we suggest that one H2-haplotype-matched self-specific Tregs derived from the transfusion blood can be activated and multiply in the host by binding to antigen-presenting cells presenting allopeptides in their major histocompatibility complex (MHC) class II (MHC-II). We also suggest that the endothelial and epithelial cells within the solid organ allograft upregulate the expression of MHC-II and attract the expanded Treg population to suppress inflammation within the graft. We further suggest that this biological process, here termed MHC-II recruitment, is a vital survival mechanism for organs  when attacked by an immune system. Today, transplantation tolerance in humans who receive an organ from a genetically disparate donor can very rarely be achieved without life-long administration of immunosuppressive therapy. While the current, non-specific immunosuppressive drugs are very good at inducing short-term graft survival, the impact on long-term engraftment has remained unchanged because most of the patients lose their transplants due to chronic rejection. Furthermore, due to general suppression of the immune system, the patients are exposed to a higher risk of infections and developing cancer. Therefore, the ultimate goal is to find a way to induce permanent donor-specific tolerance , 2.Long-term alloantigen-specific transplantation tolerance of solid organs can easily be induced in rodents without continuous supply of immunosuppressants . AlthougA widely used strategy to induce long-term transplantation tolerance in experimental animal models is to introduce the organ recipient to alloantigens in the form of blood transfusion before transplantation . MultiplSharing of a HLA/H2 haplotype between the recipient and blood donor in both humans and experimental animal models is associated with prolongation of graft survival. The effect is further optimized when the haplotype shared blood is donor specific, i.e., taken from the organ donor [donor-specific blood transfusion (DST)] –15.The mechanisms responsible for DST-induced unresponsiveness are not fully understood. However, many experimental studies in animals indicate that perhaps the most powerful mechanism involved in prolongation of allograft survival is the suppressor function of Foxp3 CD4+CD25+ regulatory T cells (Tregs). Tregs are also found to be indispensable for maintaining peripheral self-tolerance. How DST may induce Tregs has been intensively studied for decades.One central issue is whether donor-derived leukocytes are actively involved in inducing transplantation tolerance, and if so, how and to what extent. In this paper, we re-interpret an immunogenetic experiment conducted 17 years ago by Niimi et al., and we present a hypothesis that suggests that actively involved donor blood-derived Tregs and the donor organ itself are crucial for the prolongation of the allograft survival achieved in their study.via the donor organ as “passenger cells” and pretransplanted donor blood. In the early 1990s, Thomas E. Starzl and his colleagues proposed a model of microchimerism and transplantation tolerance. According to this model, allograft acceptance is the effect of a balanced double immune reaction between donor and recipient cells, which is comparable to a mutual graft-versus-host/host-versus-graft reaction, and causes reciprocal clonal exhaustion. Although the reaction eventually attenuates, it remains at a low level and keeps a dangerous increase of either of the two reactions in check. The persistence of microchimeric cells is therefore considered to be a prerequisite for long-term allograft survival in this model   . RecogniUnfortunately, this model does not explain allotolerance in mice because mice T-cells do not produce MHC-II. Therefore, a different model is required to explain similar phenomena that occur in mice.The discovery by Sakaguchi et al. in 1995 that a subset within the CD4+ T cell population that expresses CD25 are responsible for maintaining immunological self-tolerance in mice  triggerein vitro (iTregs) from the naïve CD4+ T cells population when encountering MHC/peptide complexes on dendritic cells in tolerogenic environments, such as in the presence of TGF-β and interleukine-2 (IL-2)  –34; i/p 2 (IL-2) . In heal2 (IL-2) . Tregs t2 (IL-2) , 37.foxp3-locus upstream of exon-1, also termed TSDR  Tregs show that they proliferate vigorously eostasis , 46, 47.eostasis , 48. It eostasis .In vitro studies have demonstrated that Tregs can regulate immune responses on multiple levels and via several different mechanisms. For example, they can inhibit activation and function of APCs and produce immunoregulatory cytokines, such as TGF-β and IL-10, which act directly on effector T cells by impeding their priming and effector function  are needed to induce indefinite transplantation tolerance  mice were transfused with one haplotype-matched or fully mismatched donor blood 7 days before transplantation. The recipient mice were then transplanted with a heart of different F1 mice with which they shared one haplotype  or none (groups III and VII). All combinations are illustrated in Figure  mice werTwo sets of experiments with different blood donors were conducted to ensure that the effects of pretransplant blood transfusion were not restricted to a specific strain combination. The results were similar.Optimal prolongation of graft survival was achieved in recipient mice which received one haplotype-matched DST (approximately 40% at day 100) (I). Graft survival at day 100 was also obtained in 10% of the recipients that shared one haplotype with the blood donor and the mismatched haplotype with the organ donor (IV). The survival curve in this group was quite similar to that in which the recipient and both donors shared the same haplotype but were mismatched for the other haplotype (II). However, in the latter test group, all hearts had been rejected by day 70. Recipients transfused with homozygous blood from a mismatched donor obtained moderate graft prolongation  (V). The graft was rejected immediately if the blood was derived from a haplotype-matched homozygous donor (VI). Prolongation of graft survival was also not achieved with fully mismatched DST (VII).In pilot studies with purified CD4+ T cells from mice after haplotype-matched blood transfusion, Niimi et al. found that these cells had the ability to downregulate donor-specific CTL responses in a specific way. This finding indicates that allospecific Tregs had been induced by the blood transfusion and that these Tregs were involved in prolongation of graft survival.According to Niimi et al., graft survival induced by the matched DST is probably due to priming of host T cells that recognize allopeptides presented by self(shared)-MHC indirectly on recipient APC and directly on donor APCs. However, since graft survival was also prolonged in combinations where the blood and organ donor shared only the mismatched haplotypes (IV and V), Niimi et al. suggest that presentation of allopeptides by recipient (self)-APCs is the essential pathway for tolerance induction.the organ did not have a recipient haplotype and was therefore not able to present the allopeptide.”Although indirect alloantigen recognition may have been essential to tolerance induction during the pretreatment regimen, the hypothetical-induced Tregs had to be reactivated by the transplanted organ for graft prolongation to occur. According to Niimi et al., this explains why the hearts in group III were all immediately rejected. In their own words: “… With regard to prolongation of graft survival in group II where the blood and organ donor share a recipient haplotype while being mismatched for the other, Niimi et al. suggest that the tolerance achieved is due to cross-reactivity or sharing of allopeptides between the mismatched haplotypes (haplotypes B and C).Several mechanisms may have contributed to the prolongation of graft survival obtained in the Niimi et al. study. It is, for example, well known that alloreactive T cells anergize or are deleted when reacting with donor-derived allopeptides or allo-MHC complexes in non-inflammatory transplantation settings . AlthougThe direct pathway of allorecognition is primarily associated with acute graft rejection . In expeI.via the indirect pathway of antigen recognition/presentation, i.e., recipient APCs, if (a) the donor and recipient have a sufficient match of major histocompatibility complex (MHC) classes I and II, for example, share one haplotype and (b) the recipient APCs present the donor-derived peptide that is recognized naturally by the donor-derived Treg and the indirect antigen-presenting profile (MHC/peptide profile) is functionally identical to the immunological self of the blood donor, i.e., that at least some self-peptide/MHC complexes are identical in the recipient and blood donor, the antigenic background on which that these blood donor-derived Tregs have been selected on.Whole live blood contains functional self-specific Tregs, which are transferred to the recipient by blood transfusion. These self-specific donor-Tregs can be activated  and proliferate All mice in the Niimi experiment were naive . Therefore, we also find it reasonable to assume that a substantial portion of the Tregs were self-specific tTregs, positively selected in the thymus upon encountering tissue-specific antigens before entering the periphery , 59. Becin vitro studies have shown that Foxp3 Tregs can physically out-compete non-Tregs in aggregating around APCs  responses directed against organs (either as a HVG-reaction or auto-reactive) cause the cells of the attacked organ  to produce or upregulate the production of MHC-II molecules. We have chosen to term the upregulation of MHC-II “MHC-II recruitment.” MHC-II recruitment allows for activated self-specific Tregs  to bind specifically to the organ and downregulate immune responses at the site of inflammation. This self-protecting mechanism will be activated if or when the transplanted organ is attacked by the host immune system.As indicated by the different outcomes of allograft survival in group I and, i.e., II and IV, the hypothetical multiplied self-specific donor tTregs appear to be restricted to carrying out their full regulatory potential of responses against a genetic identical organ donor. We further hypothesize thatvia rDCs before the donor organ is transplanted. The hypothetical induction of pTregs with specificity for a different MHC-II/peptide combination (D/b) than that of the blood donor-derived tTregs can explain prolongation of graft survival in group IV.The specific acquired tolerance in recipients that were transfused with one haplotype-matched DST can be explained by MHC-II recruitment by the endothelial/epithelial cells of the transplanted organ. From here, two pathways, which are not mutually exclusive, can support Tregs in performing their memory function and maintaining allotolerance: (1) semi-direct recognition of MHC-II/peptide complexes transferred directly by epithelial cells to recipient DCs (rDCs) or by epithelial exosomes and membrane vesicles –62; (2) The suggestion that MHC-II recruitment is essential for Tregs to achieve their full regulatory potential, both in transplantation tolerance and peripheral immunological self-tolerance, is consistent with the following discoveries: (1) several studies have demonstrated that endothelial and epithelial cells in human transplants and in mice upregulate their production of MHC-II molecules –66. (2) The attachment of Tregs to endothelial/epithelial MHC-II/peptide complexes may even be a short cut to reactivation of Tregs (memory Tregs) as suggested by Niimi et al. 17 years ago. Although activation of T lymphocytes is closely linked to professional APCs, recent studies have shown that hepatocytes from a transgenic mouse model, expressing stable MHC-II and featuring CD80, are able to activate CD4+ T cells. In this study, the epithelial activation of CD4+ T cells was not associated with autoimmune reactions . AdditioAlthough our hypothesis is about transplantation tolerance induced by donor tTregs, recipients of one haplotype-matched allografts may, like the donor, harbor self-specific tTregs that recognize and bind self-MHC-II/peptide complexes on the donor organ. These hypothetical recipient tTregs may have been a contributing factor to prolongation of graft survival in recipients that were not tolerated for the mismatched antigen in the donor organ, as in group II. However, since tolerance was not achieved with matched pretransplanted homozygous donor blood (A in group VI), recipient and donor tTregs appear to be incapable of inducing tolerance without the presence of an alloantigen, for example, allopeptides from shared minor antigens between the blood donor (haplotype B) and the organ donor (haplotype C) as indicated by the outcome in group II Figure .In this article, we have introduced a novel hypothesis on DST-induced transplantation tolerance based on a re-interpretation of a 17-year-old immunogenetic study by Niimi et al. . The aniBased on the immune status of the mice in the experiment of Niimi et al., two main suggestions have been presented in the hypothesis: (1) blood donor-derived self-specific tTregs can be (re)activated and multiplied by indirectly recognizing MHC/peptide complexes on host APC that correspond to the antigenic background they have been selected on in thymus; (2) the transplanted organ upregulates endothelial/epithelial expression of MHC-II if or when the organ is attacked by the host immune system. It is further suggested that the multiplied donor tTregs suppress bystander allospecific T cells and may induce infectious tolerance by binding directly to the donor organ or semi-directly to professional host APCs.This hypothesis is consistent with recent animal studies which have shown that for long-term transplantation tolerance to occur, there is a need for Tregs that recognize allopeptides presented by self-MHC-II on host and donor APCs.Several studies have indicated that tTregs constitute a more stable and mature cell population than Tregs induced from the naïve T-cell pool. Therefore, approaches that focuses on self-specific donor tTregs with specificity for self-peptides presented by shared MHC-II could be an attractive shortcut to prolongation of allograft survival because many apparently difficult steps in the generation of new Tregs from the naïve T cell pool may already have been completed. Yet, introduction of donor-specific antigens, such as donor blood and donor Tregs, prior to transplantation may increase the risk of immunizing the host.According to our hypothesis, recipients of one haplotype-matched allografts may, just like the donor, harbor self-specific tTregs that can recognize and bind to both pathways of alloantigen presentation. Expansion of such recipient tTregs could minimize the risk of immunizing the host. This mechanism may have contributed the moderate prolongation of graft survival in group II. However, it is crucial to find out why tolerance was not induced with haplotype-matched homozygous donor blood (group VI).To further support the hypothesis proposed in this manuscript, it will be important to test experimentally the longevity of Tregs alive in the recipient and whether these Tregs are essential for tolerance induction. It is also important to test whether the transplanted organ upregulates the production of MHC-II and whether these donor MHC II/peptide complexes are transferred to recipient APCs/DCs. Finally, support of the hypothesis will also require testing of whether linked suppression and infectious tolerance occurs in the donor:recipient combination.We hereby confirm that all the authors’ contribution to this manuscript meets with the criteria of authorship demanded by Frontiers.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


HER2  amplification, and other recently reported genetic alterations in sporadic PTC (sPTC) and fPTC, and assessed correlations with RTL and BRAF mutational status. We analyzed HER2 gene amplification and the integrity of ALK, ETV6, RET, and BRAF genes by fluorescence in situ hybridization in isolated nuclei and paraffin-embedded formalin-fixed sections of 13 fPTC and 18 sPTC patients. We analyzed V600EBRAF mutation and RTL by qRT-PCR. Significant HER2 amplification (p = 0.0076), which was restricted to scattered groups of cells, was found in fPTC samples. HER2 amplification in fPTCs was invariably associated with V600EBRAF mutation. RTL was shorter in fPTCs than sPTCs (p < 0.001). No rearrangements of other tested genes were observed. These findings suggest that the association of HER2 amplification with V600EBRAF mutation and telomere shortening may represent a marker of tumor aggressiveness, and, in refractory thyroid cancer, may warrant exploration as a site for targeted therapy.Extensive research is dedicated to understanding if sporadic and familial papillary thyroid carcinoma are distinct biological entities. We have previously demonstrated that familial papillary thyroid cancer (fPTC) cells exhibit short relative telomere length (RTL) in both blood and tissues and that these features may be associated with chromosome instability. Here, we investigated the frequency of  RAS and BRAF and rearrangements of RET/PTC and NTRK1, exhibit similar prevalence and distribution in both sPTC and fPTC [HAPB2 germline mutation to the predisposition to familial forms of NMTC [ALK [ETV6 [HER2 gene, reported in highly-malignant PTC nodules [HER2 amplification in PTC was observed by fluorescence in situ hybridization (FISH), and average telomere length in HER2-positive (HER2+) PTC was significantly shorter than HER2-negative (HER2−) PTC [HER2 amplification and telomere length status in 13 fPTC and 18 sPTC. Tumors were also investigated for integrity of the RET gene, which is rearranged in 10%–40% of PTC, and ALK, ETV6, and BRAF genes, which are rearranged in a minority of PTC. V600EBRAF mutation, which is associated with an aggressive biological behavior [HER2 gene intermediate amplification in fPTC compared to sPTC, and shorter telomeres in all fPTC, including those with HER2 amplification, compared to sPTC. In addition, all HER2+ samples invariably possessed the V600EBRAF mutation, but not vice versa. The association might represent a marker of tumor aggressiveness and, in refractory thyroid cancer, may indicate possible exploration for targeted therapy. Additionally, the simultaneous occurrence of these three specific molecular alterations may be suggestive of the existence of a specific fPTC subgroup.The most common histological subtype of non-medullary thyroid carcinoma (NMTC) is papillary thyroid carcinoma (PTC), which represents 75%–85% of all thyroid cancer. Although mostly sporadic (sPTC), there is some evidence for a familial form of PTC (fPTC) not associated with known Mendelian syndromes. Familial PTC is observed in approximately 5%–10% of NMTC cases , this diand fPTC . No oncoand fPTC . However of NMTC ,7,8. Gen of NMTC ,10. LessMTC [ALK  and ETV6LK [ETV6 , identif nodules , might bR2−) PTC . Of possR2−) PTC . These obehavior , was alsHER2 amplification was evaluated in 13 fPTC  and 18 sPTC . We found that isolated fPTC and sPTC nuclei were HER2− according to the Wolff criteria  examination) [HER2+ cells, ranging from 1.4% to 9% in fPTC and from 1% to 2.4% in sPTC  according to Vance [HER2 amplification in fPTC compared to sPTC (p = 0.0076)  , althoug in sPTC A,B. Baseto Vance . We foun 0.0076) C–E. We fALK, BRAF, and ETV6 genes by fluorescence in situ hybridization. We found one case of sPTC with a RET rearrangement, the remaining cases exhibited neither disruptions nor numerical changes in RET gene (see examples in We found no rearrangements of p < 0.001) (Relative telomere length was significantly shorter in fPTC samples than in sPTC samples: median = 0.93 (25th–75th percentile: 0.6–1.2) vs. 1.9 (25th–75th percentile: 1.8–2.3) for fPTC vs. sPTC, respectively (< 0.001) . This re< 0.001) .V600EBRAF mutation was detected in 9/13 (69.0%) fPTC and 14/18 (78%) sPTC (p = 0.68), which was not statistically significant. All HER2+ fPTC were V600EBRAF-positive (BRAF+), although not all BRAF+ fPTC were HER2+  [HER2 amplification (p = 0.019) in fPTC compared to sPTC. This finding indicates a degree of genetic heterogeneity in the fPTC group and suggests that HER2+ cells in fPTC possibly undergo apoptosis to a lesser extent than in sPTC. The observation adds thyroid carcinoma to the list of tumors that exhibit HER2+. HER2+ is indeed observed in a growing number of other tumors, including advanced gastric and esophageal cancer [HER2+ breast and gastric/gastroesophageal cancers more likely to respond to targeted therapies in combination with chemotherapy than HER2− tumors. A wide range of solid tumors showing deregulation of HER2 expression are regarded as biologically aggressive. Familial PTC is often associated with a more severe phenotype than its sporadic counterpart [V600EBRAF mutation. V600EBRAF is considered to have a prognostic value in PTC [V600EBRAF is also strongly associated with PTC patient mortality [HER2 amplification and V600EBRAF mutation appear to be mutually exclusive events [HER2+ fPTC bore the V600EBRAF mutation, although not all V600EBRAF-positive nodules had HER2 amplification. Of significance, none of the V600EBRAF-positive sPTC were HER2+, despite the high frequency of V600BRAF mutations in this cohort. It is not entirely clear how this discrepancy should be interpreted, considering the limited size of our cohorts. In addition, we do not know whether HER2 amplification and V600EBRAF mutation coexist in the same cells within a tumor or if they are segregated in different clones. We do not know, either, if the condition is different in our HER2+ fPTC versus sPTC with HER2+ cells <5%.Amplification of the  nodules . They obof cases ,2. Familof cases ,22, the  [HER2+)  and PTC  [HER2+) ,25, and  [HER2+) ,27. None [HER2+)  in the il cancer , ovarianl cancer , colon [l cancer , bladderl cancer . The extnterpart , and ofte in PTC , and usuortality . In conte events , here weALK, BRAF, ETV6, and RET, exhibited extensive integrity. The only case bearing a RET disruption was V600EBRAF-negative. Moreover, we found a significant difference (p < 0.001) of RTL in fPTC nodules compared to sPTC nodules, in agreement with our previous investigations [BRAF [The other evaluated genes, igations ,35. Teloigations . Dysfuncigations . Althougigations  and may igations ,40. On tigations . Recurreigations , often ins [BRAF . A subclns [BRAF . UnfortuHER2 amplification, indicating an apparently preferential association with fPTC. In this regard, the small number of patients investigated in our two cohorts might constitute a bias. Nevertheless, as a whole, the finding of V600EBRAF mutation in association with HER2+ genetic heterogeneity, short telomere length, and prevalence of multifocal tumors seems to not be a rare molecular event in fPTC and may characterize a subgroup of fPTC. The response of refractory fPTC patients of this subgroup to target therapy of trastuzumab and lapatinib should be explored.Our finding on RTL are, substantially, in keeping with the Sugishita data . HoweverThe PTCs (13 fPTC and 18 sPTC) considered in the present study were selected from the pathological files of the University of Siena. Familial recurrence of the disease was defined as the presence of at least one first-degree relative with differentiated thyroid carcinoma in the absence of any other familial syndrome. None of the fPTC patients presented with any other sporadic tumor, including breast cancer, except for one male patient with a previous squamous cell carcinoma of the auricle. The histology of all tumor samples was classified according to the World Health Organization guidelines . The fPTThe sPTC cases were from 15 females  and three males . Fourteen were classified as classical PTC, two as follicular variants, one as a diffuse sclerosing variant, and one as a trabecular variant . InformeThick sections (30 µm) were obtained from formalin-fixed paraffin-embedded (FFPE) tissue blocks of the thyroid nodules or, to assess probe cut-off, from the apparently tumor-free tissue of the contralateral lobe. Nuclei were isolated as reported , and werHER2 amplification was determined by counting the total numbers of HER2 and CEP17  signals per nucleus with a mean of 89.5 (range 34–175) nuclei. The ratio of HER2 signals to CEP17 (centromeric probe for chromosome 17) signals was calculated according to ASCO/CAP  criteria refined for breast cancer [t cancer .HER2 amplification were then re-evaluated by FISH on 4 µm histological sections to assess the distribution of possible clones. The tumor area, marked by the pathologist [Nodules with, or suspected to have, hologist , was enthologist .HER2 expression by immunohistochemistry. The expression of HER2 protein was determined using anti-c-erbB2 antibody  in accordance with the manufacturer’s instructions.Consecutive histologic sections were used to assess ALK, BRAF, ETV6, and RET rearrangements. Commercially available break-apart or single gene probes for ALK, BRAF , and ETV6  were used to verify gene integrity in nuclei isolated from FFPE tissue blocks. An arbitrary cut-off of 3% was employed, as control cells showed no split signal in 200 scored nuclei per sample. For the RET gene, a homebrew probe and a previously described cut-off value were used [Detection of ere used . Microsc® DNA Mini Kit  following the manufacturer’s instructions. RTL of sPTC was determined by quantitative PCR, carried out on 30 ng/μL genomic DNA using an MJ Mini Personal Thermal cycler  as described [36B4 gene, which encodes acidic ribosomal phosphoprotein P0, was used as the single-copy gene [V600EBRAF mutation, DNA was amplified in a final volume of 50 µL of 2× PCR Master Mix  and a final primer concentration of 200 nM. Primer sequences, PCR conditions, and interpretation of results were as previously described [DNA was extracted from fresh or FFPE tissues, using the QIAampescribed . RTL valescribed . Relativopy gene . For anaescribed .U-test  was used for statistical analysis of differences in RTL. Fisher exact test was used to compare HER2 gene amplification. All p-values were two-sided and p less than 0.05 was considered significant.Mann-Whitney t-test for independent data was performed for normal variables. To evaluate significant differences in data frequency we analyzed 2 × 2 contingency tables by the Fisher exact test. Tables with sizes larger than 2 × 2 were examined by the Chi-squared test.Epidemiological data are presented as the mean ± SD and median when necessary. The


One-quarter of neonatal deaths are attributed to infections. Maternal handwashing with soap may prevent neonatal sepsis. We examined impact of intensive handwashing promotion on handwashing behavior of mothers of neonates. In Matlab, Bangladesh, we randomly allocated pregnant women at 28–32 weeks' gestation to intensive handwashing promotion or control. Behavior change communicators used a participatory approach to motivate maternal handwashing with soap and provided soap and handwashing stations. In the neonatal period, we observed soap and water at handwashing places and, at the end of the neonatal period, we estimated impact on maternal handwashing by structured observation. Among 253 women enrolled, intervention households were between 5.7 and 15.2 times as likely as control households to have soap and water present at the handwashing station in the baby's sleeping area. Intervention mothers washed hands with soap 4.1 times as frequently as controls (95% CI 2.55–6.59); handwashing with soap at recommended times was infrequent in both intervention (9%) and control (2%) groups. Intensively promoting handwashing with soap resulted in increased availability of soap and water at handwashing places, but only a modest increase in maternal handwashing with soap. Novel approaches to motivating handwashing behavior to protect newborns should be developed and evaluated.  In 2013, an estimated 2.8 million deaths occurred in the neonatal period globally [In an observational study of 23,662 infants in Nepal, Rhee and colleagues found that neonates of mothers who reported washing their hands were at 44% lower risk of mortality than neonates of mothers who did not report handwashing , 8.In a four-arm cluster-randomized controlled trial in Pakistan investigating the neonatal mortality effects of umbilical cord cleansing with chlorhexidine and handwashing with soap promotion separately and together, Soofi and colleagues found no benefit from promoting handwashing with soap . The hanGreenland and colleagues found maternal handwashing in the neonatal period in Indonesia to be infrequent, with handwashing occurring typically because of discomfort  or disgust-related motivators (such as apparent smell) . In BangWe developed an intensive intervention to improve maternal handwashing behavior during the neonatal period, addressing several of the key barriers and motivators identified by Parveen and colleagues, described above. Our objective, in a randomized controlled trial in rural Bangladesh, was to investigate the impact of this intervention on the handwashing behavior of mothers in the neonatal period.This study was conducted in Matlab, a rural area 55 km southeast of Dhaka, Bangladesh. In 2007, 44% of mothers in the study area reported not receiving any antenatal care in the third trimester and 72% of births were attended by traditional birth attendants . The neoWoman planned to remain in the study area for at least one month before and one month after delivery.No other women living in the same household compound had previously taken part in either arm of the study.Women were not from household compounds that had participated in the qualitative research on motivators and barriers to handwashing in the neonatal period.Women were not enrolled in any other study; in some of the villages, a study on antenatal nutrition that enrolled women early in pregnancy was being implemented concurrently.Eligible women were requested to provide signed informed consent. Prior to randomization, we recorded baseline knowledge of hygiene and beliefs pertaining to childcare and neonatal illness, perceptions of risk and severity of neonatal illness, antenatal care, and demographic information, using questionnaires and observations. Staff regularly contacted women during the two weeks preceding their estimated due date to identify births as soon as possible after delivery; women and their families were also encouraged to contact study staff for birth notification.Between October 2010 and October 2011, data collectors approached primiparous women identified in the study area through the HDSS pregnancy database with a due date between December 1, 2010, and December 1, 2011. We included primiparous mothers for the handwashing study because we anticipated that they would be at a uniquely teachable moment , 16. PriWe used block randomization, using blocks of 4, to randomize participants to either the intervention or control arm. A study team member not involved in day-to-day field operations constructed the assignment table. The field team leader consulted the assignment table in order to determine the arm to which the participant was allocated. Data collectors were not blinded to the assignment status of participants, since the intervention included various hardware , which were expected to be visible during the data collector's visit to the household.We trained female behavior change communicators, who typically have Master's degrees and experience in data collection, to implement the intervention. The training consisted of didactic sessions, role plays, and field pilots and was delivered over 16 days. Both intervention and control participants received maternal and neonatal health counseling, patterned on information delivered in Projahnmo  see vis.nurture as motivator for handwashing improved convenience cues to action There is prior observational evidence supporting the beneficial effect of maternal handwashing before contact with the umbilical cord for prevention of neonatal infections [ possible pathogen transmission to the neonate for handwashing with soap: after respiratory secretion contact, before umbilical cord care, before breastfeeding, and after fecal contact. To stay consistent with typical handwashing promotion messages, we also recommended handwashing with soap before food preparation.The behavior change communication strategy was based on a theoretical model constructed a priori that included concepts of the Health Belief Model and the heuristic model for teachable moments, described by McBride and colleagues , 15, 18.fections . OtherwiWe used two principal methods to measure handwashing behavior: rapid assessments of the presence of handwashing materials and direct (structured) observations. At baseline and during postnatal days 4-5, 10–12, 13–15, and 20–22, the data collector identified the presence and location of a designated handwashing station(s), and the presence of water and soap at the handwashing station(s) . She assThe structured observation was conducted on days 30–32 postpartum and lasted 3 hours. During the observation period, a data collector positioned herself in the home/compound in order to assess handwashing behaviors at times of possible pathogen transmission to the neonate. Typically, the subject of observation was the mother; other family members interacting with the neonate  were observed as well.The primary outcome of interest was maternal handwashing with soap at times of possible pathogen transmission to the neonate. Secondary outcomes included the total number of times mothers were observed washing hands with soap during 3-hour observations; the maintenance of soap and water for handwashing anywhere in the home and specifically where newborns rest for most of the day; handwashing with soap by other household members at times of possible pathogen transmission; and the number of events observed among household members other than the mother washing hands with soap during the 3-hour observations.When this study was being planned, we were not aware of data describing the observed frequency of hand cleansing before cord cleansing. Substantially more information is available regarding the frequency of hand cleansing after defecation. Thus, to estimate the required sample size for measuring the impact of handwashing promotion on maternal handwashing behavior, we assumed that the probability of observed handwashing with soap after a fecal contact event would be 0.20 in the control arm [The primary outcome of interest was based on structured observation data and reflects the proportion of intervention-recommended times at which one or both hands were washed with soap. In order to compare the frequency of handwashing during the observation period between treatment arms, we used mixed linear regression to calculate the difference in the mean number of observed handwashing events between arms, separately among and other household members, in an intent-to-treat analysis. Although not prespecified, we also generated rate ratios to compare the proportion of intervention-recommended times at which hands were washed in the two study arms; we used log binomial regression, accounting for repeated measures at the caregiver level to adjust standard errors. We assessed differences between the study arms, with respect to observed handwashing with soap among other family members interacting with the neonate. In supplemental multivariate analyses, we adjusted for baseline differences between study arms. We also report the proportion of mothers who maintained soap and water at at least one handwashing station in the home and at a handwashing station in the room where the neonate was cared for and compare the intervention and control groups at each time point that this outcome was observed.http://www.clinicaltrials.gov (Identifier: NCT01309321). This study was reviewed and approved by the Research and Ethical Review Committees at icddr,b: International Centre for Diarrhoeal Diseases Research, Bangladesh (PR-10036).This trial was registered at Between October 2010 and October 2011, we identified 695 pregnant women reported to be between 28 and 32 weeks of gestation in the study villages . Common Participants in the two treatment arms had similar baseline measures, including demographics, soap and water at a handwashing station, wealth measures, and number of years of education for the mother . The armAt baseline, the majority in each arm, 67% of controls and 60% of intervention participants, indicated that their main handwashing station was near the tubewell. None of the participants in the control arm and only 2 (2%) in the intervention arm were observed to have their main handwashing station inside the house. Almost all participants were found to have water, but just 15 (12%) in each arm were observed to have bar soap at the main handwashing station; other types of soap were rarely observed.We documented 2 (2%) stillbirths and 4 (3%) neonatal deaths in the intervention arm and 6 (5%) stillbirths and 2 (6%) neonatal deaths in the control arm. There were no maternal deaths. All births were notified either by the respondent/family or upon telephone call by study staff following the estimated date of delivery.p < .0001)  . At all p < .0001), decaying to 51% of respondents in the intervention arm and 9% of respondents in the control arm during the last week of the neonatal period . Soap and water were observed at the handwashing station in the baby's sleeping area at 65% of intervention households and 10% of control households at the observation conducted at postnatal days 4-5  (The majority (84%) of respondents in the intervention arm and 44% of respondents in the control arm reported that the primary handwashing station used by the respondent was located in the baby's sleeping area, at postnatal days 4-5  . Across  with water alone was similar in the two groups , mothers in the intervention arm washed their hands with soap with a mean of 0.82 times (SD 1.2) during the 3-hour observation period, compared to 0.20 times by mothers in the control arm   control and 112 (90%) intervention participants . Loss to37–0.86) . During 37–0.86) . OverallHandwashing with soap was significantly more frequently observed in intervention arm household members overall and at the recommended times; however, handwashing with soap was infrequent in household members in both arms (handwashing at 6% of recommended times in intervention arm and 1% of recommended times in the control arm).At baseline, 23% of control (29/124) and 20% of intervention (25/125) participants said that another person reminded them to wash hands on a typical day. At follow-up, 28% of control arm (30/106) and 36% (40/110) of intervention said that someone reminded them. At baseline, 40% of control (50/124) and 46% of intervention (57/125) participants said they reminded someone else to wash hands on a typical day. At follow-up, 44% (47/106) in the control arm and 70% in the intervention arm (77/110) reported doing so.Intensive handwashing promotion employing emotional drivers, improving convenience, and providing visual cues led to only a modest increase in the frequency of handwashing with soap among new mothers in rural Bangladesh during the first 30 days after the birth of their child. The intervention resulted in greater maintenance of handwashing materials in locations where mothers are cocooned with neonate. However, the overall proportion of events accompanied by handwashing was low in the intervention arm compared with the times that we recommended for handwashing. Handwashing with soap was significantly higher before breastfeeding and after fecal contact among intervention mothers than among controls; however, washing with soap after fecal contact was less common than the prevalence of this behavior observed among mothers of young children in Bangladesh and elsewhere, underscoring the substantial barriers to handwashing faced by mothers of neonates .McBride et al. has described that pregnancy is a transformative moment in a woman's life, at which time she is reenvisioning her role of herself as well as creating and reacting to the expectancies of her pregnancy outcome  . Curtis We sought to increase the convenience of handwashing by providing soap and handwashing stations and encouraged maintenance of soap and water at handwashing locations throughout the newborn period. Handwashing station placement was guided by the mother and other relatives such that the mother would have access to soap and water in the key locations where she expected to spend time with the neonate. Still, the finding that only 39% of households in the intervention were found to have soap and water at a handwashing place at all five of the rapid observations suggests that maintaining handwashing materials at fixed locations at all times was difficult in the newborn period, that such maintenance had not become habitual, or that this approach to handwashing was not acceptable to our study population. Hand cleansing technology that does not necessitate as frequent replenishment may be needed to further improve hand hygiene in the newborn period.While the intervention increased the frequency of handwashing, mothers and others washed hands with soap at less than 10% of the times we recommended for handwashing. Since we completed structured observations at the end of the neonatal period during postnatal days 30–32, we cannot know whether handwashing was more common or less common during the early days of the newborn period, when babies may have been perceived to be particularly vulnerable but mothers may have been particularly fatigued or getting adjusted to the new household routine following the birth.Mothers in several low-income countries have indicated that their responsibilities increase in the postnatal period, often without increased support for household or child care activities from others . We recoIncreasing evidence regarding the role of healthy intestinal, skin, and mucosal microbiota affirms that most organisms transmitted from mothers to neonates are not pathogenic. Indeed, transfer of many commensal organisms from mother to neonate represents an important element of developing healthy microbiota and immune systems –28. TherHands get recontaminated after handwashing quickly in heavily contaminated household environments such as rural Bangladesh  and, thuWe restricted our study to primiparous mothers, since they might have been more susceptible to behavior change given their relatively larger change in self-definition, compared to multiparous mothers. However, this sample may have decreased the representativeness of our study; mothers with a newborn who also have infants or older children to care for may be less likely to change handwashing behavior because of time constraints, or because their current pregnancy does not lead to a dramatic alteration of their self-definition or role in the family and society since they already have children. We used observed handwashing as our primary outcome of interest. Persons being observed by a stranger, such as a study data collector, may alter their behavior compared to usual practice, potentially increasing their handwashing in the presence of the observer , 30. SucWe found that a handwashing promotion strategy that consisted of both providing the necessary materials for handwashing and intensive behavior change communication resulted in anemic improvements in handwashing behavior among mothers of newborns. While mothers in the intervention arm were significantly more likely to maintain needed handwashing materials in the places where they spent time with their newborns, and more frequently washed their hands, handwashing with soap was observed at a minority of the times we recommended. Promotion of handwashing with soap before umbilical cord care should be emphasized in antenatal and neonatal care messaging, given existing evidence supporting prevention of umbilical cord infections by caregiver handwashing. Understanding the extent to which hand hygiene behavior of mothers and other caregivers needs to improve at other times of potential pathogen transmission would support the development of feasible guidance on handwashing to reduce neonatal infections. Novel and feasible approaches to motivating handwashing behavior change among mothers and other caregivers to protect newborns should be developed and evaluated and, if effective, their impacts on neonatal health should be investigated.


Ventriculitis is a complication of temporary intraventricular drains. The limited penetration of meropenem into the cerebrospinal fluid (CSF) is well known. However, ventricular CSF pharmacokinetic data in patients with ventriculitis are lacking. The aim of this study was to evaluate meropenem pharmacokinetics in the serum and CSF of neurocritical care patients with proven or suspected ventriculitis.We conducted an observational pharmacokinetic study of neurocritical care patients with proven or suspected ventriculitis receiving meropenem. Multiple blood and CSF samples were taken and were described using nonparametric pharmacokinetic modelling with Pmetrics.In total, 21 patients  were included. The median (range) of peak and trough concentrations in serum were 20.16 (4.40–69.00) mg/L and 2.54 (0.00–31.40) mg/L, respectively. The corresponding peak and trough concentrations in CSF were 1.20 (0.00–6.20) mg/L and 1.28 (0.00–4.10) mg/L, respectively, with a median CSF/serum ratio (range) of 0.09 (0.03–0.16). Median creatinine clearance ranged from 60.7 to 217.6 ml/minute (median 122.5 ml/minute). A three-compartment linear population pharmacokinetic model was most appropriate. No covariate relationships could be supported for any of the model parameters. Meropenem demonstrated poor penetration into CSF, with a median CSF/serum ratio of 9 % and high interindividual pharmacokinetic variability.Administration of higher-than-standard doses of meropenem and therapeutic drug monitoring in both serum and CSF should be considered to individualise meropenem dosing in neurocritical care patients with ventriculitis. Neurocritical care patients often require implantation of an intraventricular catheter (IVC) to manage hydrocephalus and monitor intracranial pressure , 2. IVC-fT>MIC)  ≤50 ml/minute), for whom the dose was reduced to 1000 mg every 8 h at the discretion of the attending physician.min]) and after the end of the infusion (serum peak concentration [Cmax]). CSF samples (1 ml) were collected using the indwelling IVC nearest to the site of insertion (3-ml volume to sampling location) simultaneously with each blood sample just before the start of the infusion  and after the end of the infusion . Samples were centrifuged for 5 minutes at 4000 rpm immediately after sample collection and aliquoted into 2-ml propylene tubes . Aliquots were stored at −80 °C within 45 minutes after sample collection for a maximum of 4 weeks until assay. Additional data were obtained from the medical record, including weight, height, serum creatinine, bilirubin, serum C-reactive protein (CRP), serum interleukin-6 (IL-6), serum procalcitonin (PCT), serum leucocytes, CSF cells, CSF erythrocytes, CSF IL-6, CSF glucose, CSF protein, CSF drain in 24 h, Simplified Acute Physiology Score II (SAPS II), Sepsis-related Organ Failure Assessment (SOFA) score, Glasgow Coma Scale (GCS) and dexamethasone therapy.Serial blood and CSF sampling occurred for initial dose and steady state . Blood samples (4 ml) were collected using the indwelling arterial catheter just before the start of the infusion . Kcp, Kpc, Kcb and Kbc represent first-order transfer constants connecting the various compartments. The CSF compartment (X3) has an apparent CSF volume . Equation  in milligrams per hour represents the zero-order infusion of meropenem. Meropenem was cleared from the central compartment (clearance in litres per hour), which also has a volume  of the linear regression and (3) visual inspection of diagnostic scatterplots, where model predictions were generated either by the median population parameter values or by the medians of each subject’s individual Bayesian posterior parameter value distributions.The fit of the PK model to the data set was assessed in the following ways: (1) the log-likelihood value, (2) the coefficient of determination  by the total time in hours and multiplied the result by 24 to estimate the daily average AUC (AUC0–24). Penetration of meropenem into CSF was described using the CSF/serum ratio, which was calculated by dividing the CSF AUCf by the serum AUCf. Half-life was calculated using transfer rate constants. CrCL was calculated using the Cockcroft-Gault equation [Cequation . All calSimulations of 1000 patients were performed using Pmetrics to compare different dosing regimens in this study population . In addition, probability of target attainment (PTA) in CSF was analysed using Pmetrics to achieve meropenem concentrations in CSF of 1 mg/L, 2 mg/L and 4 mg/L. Linear regression was performed using Pmetrics. PTA presentation was performed using IBM SPSS software.Pseudomonas aeruginosa that was susceptible to meropenem.In total, 209 blood samples and 199 CSF samples from 21 patients were included in the model. The demographic and general clinical characteristics of patients are shown in Table max (range) was 20.16 (4.40–69.00) mg/L and the median Cmin (range) was 2.54 (0.00–31.40) mg/L. In CSF, the median Cafter 4h (range) was 1.20 (0.00–6.20) mg/L and the median Ctrough (range) was 1.28 (0.00–4.10) mg/L. The median CrCL ranged from 60.7 to 217.6 ml/minute (median 122.5 ml/minute). Individual observed meropenem concentrations and median CrCL values are shown in Table 0–24 in CSF was 26.56 mg∙h/L, and in serum it was 350.22 mg∙h/L. The values for the AUC0–24 in CSF and serum ranged from 7.44 to 85.53 mg∙h/L and from 112.95 to 768.63 mg∙h/L, respectively. The median CSF/serum ratio (range) was 0.09 (0.03–0.16). Individual AUC0–24 and penetration results are shown in Table In serum, the median C.54 0.00–1.40 mg/Lr2 of the observed-versus-predicted values   Fig. . IndividSimulated meropenem concentration–time profiles in serum and CSF of each regimen are shown in Fig. . [To our knowledge, this is the first population PK study of meropenem concentrations in serum and CSF in neurocritical care patients with ventriculitis. Furthermore, it is also the largest study investigating the penetration of meropenem into CSF. We found that meropenem poorly penetrated into CSF, with a median penetration ratio of only 9 %. However, there was considerable interindividual variability in serum and CSF concentrations and resultant CSF/serum ratios. This variation is most likely due to the range of sickness severity and the consequent effect of altered physiology on meropenem exposure, as well as to the integrity of the blood–CSF barrier. These findings are concordant with those derived from previous studies in which researchers have also described large interindividual variability in meropenem concentrations in serum  and CSF . .CSF should not be viewed as the physiological CSF volume; it is merely a scalar that explains the concentration observed in the CSF.CSF is produced by the choroid plexus . Drug peA CSF penetration of meropenem of 20 % in normal or mildly infected meninges and 39 % in inflamed meninges is described elsewhere . HoweverP. aeruginosa), Staphylococcus aureus and Staphylococcus epidermidis [Enterobacteriaceae family (<0.125 mg/L), including Klebsiella pneumoniae and methicillin-sensitive S. aureus (0.25 mg/L) [P. aeruginosa (2 mg/L) [The most dreaded pathogens in nosocomial CNS infection are aerobic Gram-negative pathogens  . However(2 mg/L) , the staThere are several limitations of this study. First, the study was relatively small, which may have hampered robust estimates of the extent of PK variability and the identification of covariates that may have explained some of the observed variance. CrCL was estimated because the measurement is not routinely performed in routine clinical care. Second, we measured total drug concentrations because protein binding is not relevant for low to moderately protein-bound drugs (unbound fraction 91–98 %) [P. aeruginosa. To address this challenge, novel dosing strategies should be investigated in further clinical studies with high daily dosages and/or with administration by continuous infusion to avoid antibiotic underexposure in the context of augmented elimination or impaired target side penetration. The safety of these higher-dose regimens must be established. An alternative approach to optimising meropenem exposure is to use individualised dosing to achieve the desired drug exposure in both serum and CSF.To our knowledge, this is the largest PK study of neurocritical care patients with proven or suspected ventriculitis. We found that meropenem showed relatively low penetration into CSF. Furthermore, high interindividual variability in serum and CSF concentrations was observed. Assuming MIC serum breakpoints, adequate CSF concentrations are not assured for pathogens with borderline susceptibility such as Currently recommended regimens for meropenem for proven or suspected ventriculitis may lead to insufficient drug concentrations in cerebrospinal fluid. Therefore, novel treatment strategies, including the possibility of therapeutic drug monitoring within serum and cerebrospinal fluid, should be investigated in further clinical studies.


In healthy CNS, A2A receptors are expressed mainly within the neurons of the basal ganglia. Alterations in A2A receptor function and expression have been noted in movement disorders, and in Parkinson's disease pharmacological A2A receptor antagonism leads to diminished motor symptoms. Although A2A receptors are expressed only at a low level in the healthy CNS outside striatum, pathological challenge or inflammation has been shown to lead to upregulation of A2A receptors in extrastriatal CNS tissue, and this has been successfully quantitated using in vivo positron emission tomography (PET) imaging and A2A receptor-binding radioligands. Several radioligands for PET imaging of A2A receptors have been developed in recent years, and A2A receptor-targeting PET imaging may thus provide a potential additional tool to evaluate various aspects of neuroinflammation in vivo. This review article provides a brief overview of A2A receptors in healthy brain and in a selection of most important neurological diseases and describes the recent advances in A2A receptor-targeting PET imaging studies.Adenosine receptors are G-protein coupled P1 purinergic receptors that are broadly expressed in the peripheral immune system, vasculature, and the central nervous system (CNS). Within the immune system, adenosine 2A (A The disd humans , 24. Howkephalin . A2A recptically  but are ptically  and can 6/2 mice . The abil cortex , 30, hipl cortex , 22, 31,l cortex –38 has ltagonism , 40. TheSCH58261  but this outflow . Similar outflow . Future 2A receptor TMSX ([7-N-methyl-11C]-(E)-8--1,3,7-trimethylxanthine) binding in the putamen was shown to be higher in PD patients with dyskinesias (disease duration: 11.1 ± 7.2 years) compared to healthy controls [11C]TMSX binding compared to healthy controls. [11C]TMSX DVR was, however, increased in the putamen in a follow-up scan after approximately a year of induction of antiparkinsonian therapy compared to the baseline scans, despite the absence of clinical dyskinesias SCH442416, and PET imaging, a significant increase was found in the binding potential in the putamen and the nucleus caudatus in PD patients with levodopa-induced dyskinesias (disease duration: 13.2 ± 5.6) compared to PD patients with levodopa treatment without dyskinesias (disease duration: 6.2 ± 3.4) and to healthy controls [PET imaging using Acontrols . On the kinesias . Similarcontrols .2A receptors in early stages of HD [2A receptors is reported in transgenic mouse models of HD [2A receptor expression in cell cultures by regulating transcription of the A2A receptor gene [2A receptor gene (ADORA2A) rs 5751876 genotype was shown to affect the age of onset of HD in humans [2A receptors rescues cognitive performance impaired by the disease [2A receptor agonists on the other hand have shown to reverse motor deficits [ In vivo A2A receptor-targeting PET imaging using [11C]KF18446 has been used to demonstrate reduced A2A receptor expression in an animal model of HD TMSX have demonstrated that A2A receptor expression is increased in the NAWM of patients with SPMS compared to age- and sex-matched controls  associates with decreased susceptibility risk of MS . Im. Im2A re and PET . The ideironment , 65, 102ironment , 104. Mosk of MS  and withsk of MS , also su2A receptor antagonists such as caffeine or SCH58261 has been shown to significantly reduce clinical scores in multiple mice and rat models of EAE β-CIT-SPECT (single-photon emission computed tomography) can be used for imaging dopamine transporter availability for differential diagnostics of early or atypical PD, [11C]PIB for identifying amyloid pathology in early AD if routine morphological imaging is normal, and [18F]FDG (2-deoxy-2-[fluorine-18]fluoro-D-glucose) for detecting hypometabolism and differentiating dementia with Lewy bodies (DLB) or frontotemporal lobe degeneration (FTD) from AD. In addition, [123I]β-CIT-SPECT may aid in differentiating between DLB and AD. For imaging neuroinflammation, [18F]FDG could theoretically be used for detecting hypermetabolism, but due to its unspecificity, it is of limited value in clinical practice compared to routine MRI imaging and cerebrospinal fluid (CSF) analyses. Thus, when imaging the detailed mechanisms of A2A receptors in neuroinflammation, more specific probes, such as A2A receptor-binding radioligands, are needed.PET imaging of A in vivo PET studies demonstrate greatest A2A receptor ligand binding in the basal ganglia, whereas low radiotracer accumulation was shown in cortical areas and cerebellum TMSX, [11C]Preladenant, [11C]SCH442416, [18F]MNI-444, and [11C]KW6002, have been tested in human subjects. Their chemical structures are presented in In the healthy CNS, humanrebellum , 129–131 binding . Regardied above , 16, 13311C]TMSX is a methylxanthine analog of KF17387. It is the most widely used A2A receptor radioligand and its binding to A2A receptors in humans has been described in the brain [11C]TMSX (previously named KF18446) was first developed by Ishiwata et al. [2A selectivity over A1 and high nonspecific binding [11C]TMSX shows relatively low affinity for the A2A receptor  by approximately 5% when compared with metabolite corrected plasma input [11C]TMSX has been developed and validated [11C]TMSX photoisomerization.Acquiring metabolite corrected plasma input function via arterial cannulation and repeated arterial sampling for the measurement of the radioligand activity and metabolism is considered the golden standard in brain PET image analyses especially with novel ligands without a priori knowledge of the ligand's kinetics and metabolism. However, this methodology can be unpleasant for the study subjects, may be prone to errors, and requires more expert personnel. Therefore, optional methods for obtaining plasma input function have been developed, including independent component analysis  and intema input . Consequalidated . Dosing 11C]SCH442416 (5-amino-7-(3-(4-[11C]methoxy)phenylpropyl)-2-(2-furyl)pyrazolo-1,2,4-triazolopyrimidine) was the first suitable nonxanthine radioligand for the imaging of A2A receptors. In a blocking study with vipadenant (an A2A receptor antagonist), the highest radioligand binding measured as metabolite corrected VT was seen in putamen (VT ~ 0.6 ml/cm3), followed by caudate, nucleus accumbens, thalamus, and cerebellum (VT ~ 0.3 ml/cm3) [2A receptor blocking with vipadenant resulted in notable 3-4-fold reduction in total [11C]SCH442416 binding (VT) in striatal ROIs and also in about up to 2-fold reduction in cerebellum. Two later studies have shown very different specific binding potentials in the putamen when using the cerebellum as a reference region for the estimation of specific radioligand binding. Grachev et al. Preladenant has high affinity for the A2A receptor and >1000-fold selectivity to the A2A over the other adenosine receptor subtypes [11C]Preladenant was recently published [11C]Preladenant has a DVR of 7.9 ± 2.3 in the putamen and shows lower binding in the frontal cortex, thalamus, and cerebellum [11C]Preladenant reduced striatal binding to extrastriatal levels but also reduced extrastriatal binding MNI-444 is the only [18F]-labeled A2A radioligand used in humans. It has relatively high affinity (Ki = 2.8 nM) for the human recombinant A2A receptor KW6002 shows high binding in the striatum, but binding is also detected in the cerebellum and thalamus. In addition, preblocking with A2A receptor antagonist KW6002 reduced [11C]KW6002 binding to A2A receptors in all studied brain regions KW6002 binding was found TMSX binding occurs in putamen and caudate, there appears to be some specific, albeit lower, A2A receptor binding in extrastriatal tissues such as cortical gray matter and cerebellum. The rate of specific binding, calculated as BP/VT, has been reported to be as high as 53% in cerebellum and 37.8–42.7% in cerebral cortex for [11C]TMSX TMSX based on the same algorithm used for [11C]PK11195 studies (SuperPK software) [Even though the highest specific [11C]TMSX . Similaroftware) . Importaoftware) .2A receptor antagonists and agonists in a variety of neurological conditions. A2A receptors are ubiquitously expressed in various areas of the CNS, but their significance in the context of the different CNS diseases still needs clarification. Pathological processes in CNS diseases are particularly difficult to investigate for reasons such as the difficulties in obtaining representative biopsies from the brain. PET imaging, on the other hand, provides an excellent opportunity to evaluate disease-specific pathology in vivo, by allowing quantitative study of the receptors of interest in an appropriate pathological environment in situ. With the increasing variety of A2A receptor-binding PET ligands available for use in human in vivo PET imaging, there is good likelihood that PET imaging will improve our understanding of the involvement of A2A receptors in the pathophysiology and pathogenesis of brain diseases, both in the neuronal compartment of the basal ganglia and in relation to inflammation, such as in progressive MS. Groups of patients can be studied cross sectionally at various stages of a given disease or, alternatively, PET imaging can be applied longitudinally to evaluate alterations in the A2A receptor in the course of the disease or in response to treatment. PET imaging of neuroinflammation has relied heavily on TSPO-binding radioligands, but methodological challenges related to TSPO-imaging has directed the field to actively seek alternative imaging probes. A2A receptor PET imaging provides one such alternative that is worth further exploring.There is increasing interest in the therapeutic development of A