There is evidence that the neural response to increasing working memory (WM) load is modulated by age and performance level. For a valid interpretation of these effects, however, it is important to understand, whether and how they are related to gray matter atrophy. In the current work, we therefore used functional magnetic resonance imaging (fMRI) and voxel-based morphometry (VBM) to examine the association between age, performance level, spatial WM load-related brain activation and gray matter volume in 18 younger high-performers (YHP), 17 younger low-performers (YLP), 17 older high-performers (OHP), and 18 older low-performers (OLP). In multiple sub regions of the prefrontal cortex (PFC), load-related activation followed a linear trend with increasing activation at increasing load in all experimental groups. Results did not reveal differences between the sub groups. Older adults additionally showed a pattern of increasing activation from low to medium load but stable or even decreasing activation from medium to high load in other sub regions of the PFC (quadratic trend). Quadratic trend related brain activation was higher in older than in younger adults and in OLP compared to OHP. In OLP, quadratic trend related brain activation was negatively correlated with both performance accuracy and prefrontal gray matter volume. The results suggest an efficient upregulation of multiple PFC areas as response to increasing WM load in younger and older adults. Older adults and particularly OLP additionally show dysfunctional response patterns  in other PFC clusters being associated with gray matter atrophy. Aging is associated with cognitive changes . HoweverDecreasing WM performance with advancing age is reflected by functional cerebral changes particularly involving prefrontal brain regions . The kinReasons for these apparently inconsistent interpretations may be found in differing study designs . Moreover, the meaning of activation differences between older and younger adults is highly dependent from other factors such as performance accuracy. In fact, prefrontal hyperactivation or increased bilaterality in older adults together with equal or higher performance accuracy compared to younger adults were consistently attributed to successful compensation . In thisAnother factor modulating age-related activation differences is WM load. In fact, pronounced age effects were found by paradigms in which task demands were experimentally manipulated . Higher The most popular aging model considering both performance accuracy and task load is the Compensation-Related Utilization of Neural Circuits Hypothesis (CRUNCH). The CRUNCH model was published by Noteworthy, an alternative option to examine the association between WM performance and brain activation is the comparison between high- and low-performers. FMRI results of Bauer and colleagues, for example, indicated that younger and older high-performers showed similar patterns of increasing prefrontal activation with increasing WM load . This paA second aim addressed the association between age, performance level and changes in gray matter. Gray matter alterations in older individuals were reported by several studies and can be reflected by cortical thinning, a decreased brain tissue surface or a reduction of brain volume after the second decade of life . ReasonsGray matter volume loss does not affect all brain regions to the same extent. Whereas gray matter volume of parahippocampal, cingulate or occipital brain structures appears to be relatively preserved from age-related atrophy , gray maTaken together, there is much evidence regarding age-related changes in cognition, brain structure and cerebral activation. However, there is sparse evidence for how these different factors are related to each other. Specifically, it remains unclear whether altered load-related prefrontal activation patterns in older adults are linked to age-related prefrontal gray matter atrophy or whether these changes are independent processes manifesting at higher ages.In the current experiment, we therefore used fMRI and voxel-based morphometry (VBM) to examine the association between performance level, spatial WM load-related brain activation and prefrontal gray matter volume in different age cohorts.Based on the previous theoretical considerations, younger age and higher performance level should be associated with an efficient upregulation of prefrontal cortex activation as neural response to increasing WM load. Particularly in younger individuals and older high-performers, we therefore expect an increase of prefrontal activation from the lowest to the highest load level (positive linear trend) as quantified by fMRI. Specifically, we expect increased linear trend related activation in younger compared to older adults and in older high-performers compared to older low-performers. In older low-performers, by contrast, we particularly expect increasing load-related activation from low to medium task load but stable or decreasing load-related activation from medium to high task load as indicated by quadratic trends. These quadratic trends probably reflect dysfunctional neural responses. Consequently, older low-performers should show increased quadratic trend related activation compared to older high-performers. Moreover, we expect increased quadratic trend related activation in older compared to younger adults.Regarding structural differences, we expect lower gray matter volume in older compared to younger individuals and in older low-performers compared to older high-performers in dorsolateral and ventrolateral prefrontal brain regions as quantified by VBM.Finally, the extent of quadratic trend related activation should be negatively correlated with prefrontal gray matter volume and performance accuracy in older low-performers, indicating a neural dysfunction being associated with structural atrophy.Table 1): younger high-performers (YHP), younger low-performers (YLP), older high-performers (OHP), and older low-performers (OLP). Sample size was determined by an a priori power analysis using G∗Power 3.1 .The study included 70 healthy volunteers between 20 and 80 years of age. Participants were divided into two groups of 35 younger and 35 older individuals. To analyze the impact of performance level, both age groups were further subdivided into high-performers and low-performers by median split , eventually resulting in four experimental groups  . Particit(33) = 2.28; p = 0.029] and the MWT score . OHP and OLP did not differ with respect to school education, MoCA score and MWT score, but with respect to age . However, age distribution in both OHP and OLP was rather bimodal and variances were different . Accordingly, a median test was computed not indicating different medians .Next to the MoCA score, we collected the years of school education and scores in the multiple choice vocabulary test (MWT)  to estimFigure 1). Each participant performed four trials per CBT sequence length and eight baseline trials. The different experimental conditions were counterbalanced with the same sequence for all participants.To assess spatial WM, we used a modified version of the Corsi-Block-Tapping test (CBT) . The CBTEach experimental trial consisted of an encoding phase (in which participants were instructed to learn the presented sequence), a maintenance phase (delay period), and a retrieval phase (during which participants were instructed to reproduce the presented sequence).The encoding phase started with the onset of the first target block of every sequence and ended after the presentation of the last one. Duration of each target block and inter-stimulus intervals between the blocks were 1000 ms. Hence, in dependence of the respective load level, the encoding phase duration varied between 7000, 9000, and 11.000 ms.After the encoding phase, the maintenance phase (delay period) started. Duration of the maintenance phase was set to 1500, 2000, or 2500 ms  in order to improve the event-related sampling quality and efficiency of the design. During the maintenance phase, only the four horizontal blocks were shown and participants were required to keep in mind the sequence presented before.After the maintenance phase, the retrieval phase started with the instruction “Press now” at the bottom of the screen. During the retrieval phase, participants were required to reproduce the sequence of target blocks presented before in the correct temporal order by performing sequential button presses on a keypad with four horizontally arranged buttons. Each of these four buttons represented the corresponding block on the screen. A direct feedback was given confirming each button press by a change of the of the respective block’s color. The retrieval phase lasted until the last button press (but with a maximum length of 20.000 ms). Overall, each participant performed 20 trials with a duration of about 10 min.Before the experiment, participants were instructed to memorize the correct locations and temporal order of the presented target blocks. For retrieval, participants were advised to reproduce the presented target sequences by successive button presses and to respond as fast and as accurate as possible. Additionally, participants performed a 2-min series of practice trials on a PC outside the scanner, including two load c and one load 5 trial.The four horizontally arranged black blocks (RGB 0 0 0) were displayed on a gray background (RGB 163 163 163). Target blocks were displayed in red (RGB 255 0 0). Yellow color after button press indicated the given response (RGB 255 255 0).∗-weighted echo planar imaging (EPI) sequence . Via a dual-mirror mounted to the head coil, participants saw the visual stimuli presented on a monitor near the end of the tube. Before the EPI sequence, field map sequences were applied to control for magnetic field inhomogeneities. T1-weighted structural images were assessed with 160 sagittal slices of 1 mm slice thickness using a magnetization prepared rapid gradient echo (MPRage) sequence . Time of acquisition in the scanner was approximately 20 min per participant.A three Tesla Siemens Magnetom Verio Scanner was used for data acquisition. Functional images were recorded using a T2post hoc comparisons. Demographic group differences of interest  were analyzed using two-sample t-tests. SPSS Statistics 22 was used for behavioral data analyses. Significance level was set to α = 0.05, two-tailed.To analyze the impact of load and group on the number of CBT errors, we computed a 4  × 4  repeated measures ANOVA including Bonferroni-tests for 1 was used running under MATLAB R2016a . To account for the time needed for the magnetic field to become a steady state, the first three images of every EPI-recording session were discarded. Preprocessing of the EPI images included unwarping and realignment to the first volume (b-spline interpolation), slice time correction, normalization to the standard space of the Montreal Neurological Institute (MNI) brain, and smoothing with an isotropic three-dimensional Gaussian kernel with a full-width-at-half-maximum (FWHM) of 9 mm. Data were analyzed using a general linear model (GLM) with four regressors for the encoding phase , one regressor for the maintenance phase, and four regressors for the retrieval phase . All instead of only correct trials were included into the design to avoid type 1 error due to different trial numbers at the different load levels d levels . AlthougPrevious findings revealed that age-related changes within memory seem to particularly affect the acquisition and early retrieval of new information , which wWe built contrasts for positive linear (-3/-1/+1/+3) and quadratic (-1/+1/+1/-1) trends. Theoretically, positive linear trends reflect continuously increasing activation with every higher load level indicating an efficient upregulation of neural activity at increasing load. Quadratic trends, by contrast, reflect an increase of activation from load c to load 4/5 and a decrease from load 4/5 to load 6 suggesting a resource ceiling at load 4/5 . Noteworthy, activation patterns that do not strictly follow the described patterns can also reach significance in trend analyses: A positive linear trend, for example, may be characterized by increasing activation from load c to load 4 with no further increase from load 4 to load 5 or 6. This pattern would rather indicate a neural resource ceiling at load level 4 than an efficient upregulation. Similar variations can be observed for quadratic trends and there may even be activation patterns that follow both linear and quadratic functions. Consequently, we plotted mean contrast estimates for the different load levels and experimental groups separately to be able to specify the linear and quadratic trends identified by our analyses.t-tests for each group separately. Data were analyzed at whole-brain level and by a region of interest (ROI) approach, both on the voxel level with a significance threshold of p < 0.05 and a family-wise error (FWE) correction for multiple comparisons. Differences between older and younger adults and between OHP and OLP regarding linear and quadratic trend related neural activation were analyzed using two-sample t-tests. Again, data were analyzed at whole-brain level and by a ROI approach, both on the voxel level with a significance threshold of p < 0.05 and an FWE correction for multiple comparisons.Neural activation patterns associated with linear and quadratic trends were analyzed using one-sample a priori defined ROIs included different dorsolateral and ventrolateral PFC sub regions, including Brodmann areas (BAs) 9 and 46 as well as 44, 45, and 47, respectively. For this, we used the corresponding ROI masks of the automated anatomical labeling atlas (AAL)  in older adults (see Introduction), as (AAL) , implemeas (AAL) .α = 0.05, two-tailed.To be able to draw conclusions about the meaning of quadratic trend patterns for behavioral performance , the association between the extent of quadratic trend related brain activation and performance accuracy in OHP and OLP was examined by calculating bivariate Pearson correlations between the contrast estimates in the respective significant peak voxels and the total number of CBT errors. Significance level was set to 2 in SPM123 running under MATLAB R2016a . Before preprocessing, all structural images were inspected for artifacts and the origin of each image was set at the anterior commissure.For VBM analyses, all MR structural image data were processed using the CAT12 toolboxFor preprocessing, T1 weighted images initially were spatially registered and segmented into gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF) using the tissue probability maps (TPM) provided by SPM12. This procedure resulted in modulated normalized volumes reflecting the relative differences in regional GM volume. After preprocessing, a quality check was done by visual inspection of the resulting files and by positively checking the sample homogeneity using the implemented tool in CAT12. Finally, data were smoothed with a kernel of 8 mm in SPM12. Total GM volume as well as total intracranial volume (TIV) consisting of GM, WM, and CSF were estimated per subject for statistical analysis.t-tests with a peak voxel significance threshold of p < 0.05 and an FWE correction for multiple comparisons (voxel level). Significant voxels of the whole-brain analysis were labeled using the Adult brain maximum probability map . GM volume differences between younger and older adults and between OHP and OLP were analyzed at whole-brain level and in different prefrontal ROIs using two-sample p < 0.05 at the voxel level with an FWE correction for multiple comparisons.In addition, we conducted multiple regression analyses including both functional and structural MRI data. Prefrontal GM volume was included as covariate of interest to analyze the relationship between the extent of the positive linear or quadratic patterns and prefrontal GM volume in the group of older individuals and the sub groups of OHP and OLP. To estimate GM volume in dorsolateral and ventrolateral PFC sub regions, the respective ROIs  were coregistered on the preprocessed GM images . Following this, the averaged GM volume of this mask was readout for each subject and included in the statistical analysis described above. Functional ROIs were tested at a significance threshold of Figure 2) revealed significant main effects of group  and load , as well as a significant group x load interaction effect . Post hoc Bonferroni-tests indicated significant differences for all groups and all load levels (p < 0.05). The results indicate a disproportionally increasing number of errors with increasing load in all groups. OLP made the most errors, followed by YLP, OHP, and YHP. Moreover, the load-related increase of error rates varied between the groups.The 4  × 4  repeated measures ANOVA for the number of errors .Results did not reveal significant positive trends at whole-brain level for OHP and OLP. For YHP, positive trend related activity was found within bilateral superior frontal gyrus and left inferior parietal lobule. For YLP, positive trend related activity was found within bilateral superior frontal gyrus .Region of interest analyses revealed significant positive trends for all four groups in different sub regions of the dorsolateral (BAs 9 and 46) and the ventrolateral  PFC. These trends indicate an efficient upregulation of neural activity with increasing task load , OLP showed various quadratic patterns in multiple dorsolateral and ventrolateral PFC sub regions . A comparison of the different trends in the four experimental groups is illustrated in Figure 5A.Region of interest analyses showed that only older adults showed quadratic trend related brain activation in different PFC sub regions at Appendix, Table A2), direct group comparisons between older and younger adults revealed that older adults showed stronger quadratic trend related activity than younger adults within different anterior and posterior brain regions . ROI analyses additionally showed increased quadratic trend related activation in older compared to younger adults  within several dorsolateral and ventrolateral PFC sub regions .At whole-brain level .Corresponding differences between OHP and OLP did not reach statistical significance at whole-brain level. ROI analyses, however, indicated stronger quadratic trend related activity in OLP compared to OHP within left BA47 of the ventrolateral PFC .Finally, the extent of the quadratic trend was associated with lower CBT performance accuracy in OLP, but not in OHP: Activation intensity in two of the respective peaks was positively correlated with the total number of CBT errors. Both of these peaks were located in the left dorsolateral PFC .Whole-brain analysis revealed less GM volume in older compared to younger adults in various brain regions including different parts of the PFC .Comparing OHP with OLP, whole-brain analyses yielded no significant differences. ROI analyses, however, indicated less GM volume in OLP than in OHP within left BA 46 of the dorsolateral PFC .Regression analyses did not reveal negative correlations between prefrontal GM volume and the extent of the positive linear trend for older adults. By contrast, we found negative correlations between GM volume of different dorsolateral and ventrolateral PFC sub regions and the extent of the quadratic trend for older adults, as well as for OLP and OHP separately  indicating an efficient recruitment of neural resources as response to increasing WM demands, irrespective of age and performance level. Older individuals, however, additionally showed a pattern of increased activation at medium compared to low task load but stable or decreased activation at high compared to medium task load in other prefrontal sub regions as being indicated by quadratic trends. Older low-performing subjects showed this response pattern in multiple dorsolateral and ventrolateral prefrontal sub regions. The extent of this pattern was associated with reduced performance accuracy and lower prefrontal gray matter volume suggesting a regional limitation of neural resources being associated with structural deficits. The present results point toward age-related prefrontal cortex atrophy associated with possibly dysfunctional activation patterns. This reduction may first involve some dysfunctional clusters while others are unimpaired.In line with previous findings , 2014, bFunctional imaging data analyses revealed neural activation changes associated with increasing WM load in all experimental groups. Younger high-performers, younger low-performers, older high-performers and older low-performers all showed load effects in different prefrontal parts of the WM network. Indeed, all experimental groups showed an upregulation (positive linear trends) of several dorsolateral and ventrolateral prefrontal regions, indicating an efficient recruitment of neural resources as response to increasing task load. Contrary to our hypotheses, however, there were no group differences in the extent of neural upregulation, neither between younger and older adults nor between high- and low-performing seniors.Importantly, only older individuals additionally showed quadratic trend patterns over the statistical threshold  which may reflect a recruitment of neural resources at lower task demands but exhausted neural resources at higher demand levels. In line with these findings, older individuals showed more quadratic trend related activation than younger individuals in multiple sub regions of the prefrontal cortex.Whereas older high-performers showed this pattern in one cluster located in left dorsolateral prefrontal cortex, these patterns were more frequent in older low-performers  affectinTaken together, the results suggest specific prefrontal functional alterations with advanced age. Younger individuals show an efficient upregulation of prefrontal cortex activation to meet the requirements of increasing task load. Older individuals show similar patterns in several prefrontal sub regions but, in contrast to younger individuals, additional possibly dysfunctional patterns in other prefrontal cell clusters. Number and extent of these patterns appear to be lower in high-performing than in low-performing seniors, probably indicating a more preserved prefrontal functional integrity in the former group.As expected, VBM analyses revealed reduced gray matter volume of multiple brain regions in older compared to younger individuals including pronounced differences in frontal areas. These findings are in line with previous research indicating age-related regional cortical atrophy and a specific structural vulnerability of the prefrontal cortex . MoreoveMost importantly, the results of the current work revealed a direct association between neural activation and structural integrity in older adults. As mentioned above, older adults – particularly older low-performers – showed possibly dysfunctional neural response patterns in some prefrontal sub regions as being indicated by quadratic trends. These response patterns were characterized by an increase of prefrontal activation from low to medium task load but no further increase or even a decrease of activation from medium to high task load. Our findings show that the extent of this pattern was inversely associated with regional dorsolateral and ventrolateral prefrontal gray matter volume among older individuals indicating that the dysfunctional recruitment of resources at increasing task load in older adults is related to reduced gray matter volume in the respective brain regions. Noteworthy, this structure-function relationship was found for both low- and high-performing individuals, indicating a general association between prefrontal cortical atrophy and neural prefrontal dysfunctions at higher ages. Older high-performers, however, showed the described dysfunctional neural response pattern in one cluster of the prefrontal cortex whereas this pattern was found in multiple prefrontal sub regions in older low-performers which may point toward a more preserved functional cerebral status in high-performing seniors. Confirming this assumption, quadratic trend associated activation was lower in older high-performers than in older low-performers and negatively correlated with performance accuracy only in the latter group.In conclusion, the current work provides important information about the association between age, performance level, WM load-related neural activation and gray matter integrity. Our results suggest an upregulation of prefrontal cortex activation in response to increasing WM demands in high- and low-performing younger and older individuals as reflected by increasing activation in multiple sub regions of the prefrontal cortex. Whereas results yielded no differences between the sub groups regarding this upregulation, older individuals additionally showed dysfunctional neural response patterns in other prefrontal regions, which may reflect a recruitment of neural resources at lower task demands but exhausted neural resources at higher demand levels . In older low-performers, these patterns were associated with both reduced performance accuracy and gray matter atrophy in different sub regions of the prefrontal cortex probably reflecting a regional limitation of neural resources associated with prefrontal structural atrophy. In older high-performers, number and extent of dysfunctional clusters was lower than in older low-performers indicating that the cognitive status of a person does not only seem to be determined by age but particularly by the functional status of the brain. In fact, the current findings support the idea of higher brain reserve in high- compared to low-performing seniors. Brain reserve is defined as an increased adaptive neuroplasticity  due to lNoteworthy, older high- and low-performers differed with respect to mean age, although age was controlled in most analyses. However, even if age differences should have attenuated some of the described effects, this only confirms that higher age is associated with increased cognitive as well as functional and structural cerebral dysfunctions. Either way, the interpretation of results does not seem to be sophisticated. Future research endeavors should focus on a further connection between cognitive, functional and structural data. The association between the current results and white matter integrity, for example, might be of particular interest. In fact, some studies revealed that the structural integrity of frontal fiber tracts was related to prefrontal cortex activation and cognitive performance . Only thAll authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication.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.https://www.frontiersin.org/articles/10.3389/fnagi.2018.00265/full#supplementary-materialThe Supplementary Material for this article can be found online at: Click here for additional data file.


Human cytomegalovirus (HCMV) causes a ubiquitous infection which can pose a significant threat for immunocompromised individuals, such as those undergoing solid organ transplant (SOT). Arguably, the most successful vaccine studied to date is the recombinant glycoprotein-B (gB) with MF59 adjuvant which, in 3 Phase II trials, demonstrated 43–50% efficacy in preventing HCMV acquisition in seronegative healthy women or adolescents and reduction in virological parameters after SOT. However, the mechanism of vaccine protection in seronegative recipients remains undefined.We evaluated samples from the cohort of seronegative SOT patients enroled in the Phase II glycoprotein-B/MF59 vaccine trial who received organs from seropositive donors. Samples after SOT (0–90 days) were tested by real-time quantitative PCR for HCMV DNA. Anti-gB antibody levels were measured by ELISA. Neutralization was measured as a decrease in infectivity for fibroblast cell cultures revealed by expression of immediate-early antigens.Serological analyses revealed a more rapid increase in the humoral response against gB post transplant in vaccine recipients than in those randomised to receive placebo. Importantly, a number of patient sera displayed HCMV neutralising responses – neutralisation which was abrogated by pre-absorbing the sera with recombinant gB.We hypothesise that the vaccine primed the immune system of seronegative recipients which, when further challenged with virus at time of transplant, allowed the host to mount rapid immunological humoral responses even under conditions of T cell immune suppression during transplantation. Evidence before this study:Our understanding of HCMV vaccine mediated protection in seronegative solid organ transplant recipients is limited. Attempts to identify protective immunological mechanisms have focused on characterisation of immune responses immediately following vaccination. The analyses so far have failed to provide evidence for protection being mediated by conventional humoral mechanisms such as neutralization or activation of antibody dependant cellular cytotoxicity.Added value of this study:We present, for the first time, analyses of the post-transplant immunological responses of vaccinated individuals after they had been challenged with the virus at the time of transplant. The results are consistent with the gB/MF59 vaccine priming the pre-transplant immune system of seronegative recipients because, upon challenge with the virus, vaccinees rapidly generated an elevated gB antibody response that included classical neutralizing activity. Importantly, the response was greater than that seen in recipients of placebo.Implications of all the available evidence:These studies highlight the importance of studying immune responses beyond the immediate post vaccination phase. In practice, solid organ transplantation provides a tractable human challenge model for HCMV and shows how pharmacodynamic assessment of candidate vaccines may potentially identify correlates of immune protection. We recommend that this extended study design is considered when novel HCMV vaccines are evaluated in the future with separate analyses of initial immune priming and immune response to subsequent viral challenge.Ilona Baraniak: Data curation, Formal analysis, Writing - original draft. Ariane C. Gomes: Data curation, Formal analysis, Writing - original draft. Isabella Sodi: Data curation. Toby Langstone: Data curation. Emily Rothwell: Data curation. Claire Atkinson: Data curation. Sylvie Pichon: Writing - original draft. Fabienne Piras-Douce: Writing - original draft. Paul D. Griffiths: Formal analysis, Writing - original draft. Matthew B. Reeves: Formal analysis, Writing - original draft.Alt-text: Unlabelled box1As with all members of the herpesvirus family, HCMV causes a lifelong, persistent infection in its host. Infection with HCMV is common with sero-prevalence ranging from 45% to 100% de novoArguably, the most successful vaccine studied so far is the recombinant subunit glycoprotein-B with MF59 adjuvant. Phase II clinical trials with seronegative post-partum and adolescent women showed 43–50% reduction in HCMV acquisition among the vaccinated group Here we extend our studies to include serum samples collected post-transplant. In this study we focus on the D+/R- cohort (donor (D) organ from a CMV seropositive individual and recipient (R) CMV seronegative) recruited to the original phase II trial to investigate this. We report that vaccination with gB/MF59 primes the immune system of seronegative recipients to mount larger immunological responses in comparison to recipients of placebo once challenged with the virus, even under conditions of immune suppression during transplantation. Crucially, this boosting of the gB antibody response revealed evidence of gB-specific neutralizing antibodies which were not detected in a prior study of sera post-vaccination but pre-transplant 22.1The study was approved by the Research Ethics Committee and all patients whose samples were investigated here gave written informed consent 2.2The population from whom samples have been evaluated and described in this work is the highest risk cohort of seronegative solid organ transplant patients who were enroled in the Phase II randomised and double-blinded placebo controlled cytomegalovirus glycoprotein-B vaccine with MF59 adjuvant trial and received organs from seropositive individuals, described elsewhere 2.3ELISAs for total gB have been described previously ,20. Brie2.4Serological analyses were performed using commercially available line immunoassay kit recomLine CMV IgG and recomLine CMV IgM (Mikrogen Diagnostik) according to manufacturer's instruction. This stripe test contains recombinant antigens to detect IgG and IgM antibodies directed against HCMV from human sera. The antigens included in the test were IE1 (IE1 protein); CM2 ; p150 ; p65 ; gB1  and gB2 .2.5To assess sera for neutralizing capacity, HCMV Merlin was pre-incubated with sera or ITC88 antibody ,22 for 1For the pre-absorption experiment, serum samples (1/10) were pre-incubated with 100 µg/ml of gB vaccine antigen (Sanofi Pasteur) for 1 h at 37 °C prior to incubation with virus.33.1Previously, we reported that gB vaccination reduces the incidence of viraemia in our transplant cohort particularly in our D+R- group. To investigate the clinical impact of gB vaccination in more detail A we anal3.2n = 4) three had gB antibody levels below the negative cut off baseline throughout most of their post-transplant period and only one placebo patient developed detectable anti-gB antibodies early post-transplant (n = 10) in this high risk D+R- group was different. On average, vaccinated individuals produced detectable responses against gB more quickly than did placebo recipients once challenged with the virus. Indeed, increased IgG responses were detectable within 7 days of transplant in the majority of vaccinees . Similarly, no IgM responses were detected in the majority of vaccinated D+R- patients at days 7 and 35 post transplantation. However, two vaccine patients did have detectable IgM responses to HCMV antigens other than gB (Table S1B).3.4The data thus far argued for a vaccine dependant boost in gB antibody levels post challenge with HCMV (i.e. post-transplant). The key question, however, was whether this boost in gB antibody levels may reveal a hitherto undetectable response in the pre-transplant sera that could explain vaccine efficacy. Thus we asked whether the increase in gB IgG levels early post-transplant revealed evidence of neutralizing activity previously undetectable in the pre-transplant sera samples Interestingly, the analysis of sera from seronegative vaccine recipients in the post-transplant period showed evidence of neutralizing antibody activity as soon as 7 days post-transplant and the majority of those analysed had antibodies that were able to neutralize infection at day 35 post-transplant , with vaccine antigen recombinant gB should reverse the neutralisation. To do this, we selected the serum samples with the highest neutralising response  and test4Despite important studies spanning several decades of HCMV vaccine research, there is still no HCMV vaccine licensed for clinical practice. An important feature of HCMV that may be impacting on progress is the natural history which, arguably, is much more complex than viruses that have previously been controlled by immunization to prevent primary infection A major correlate of protection of many successful vaccines has been neutralizing antibodies To build on this insight, we took advantage of the unique nature of the transplant model – which effectively is a human challenge model with a defined time-point of HCMV infection post vaccination Although the sample sizes were small, the data herein show that vaccinated patients did not experience subsequent episodes of viraemia. This suggests that these patients gain control of the virus much quicker post-transplant. We hypothesized that the control was driven by adaptive immune responses against gB and, particularly in the early times post-transplant, could be linked with the humoral response given that these patients are T cell immune-suppressed. Consistent with this, a rapid increase in the level of anti-gB antibodies post-transplant was observed in some vaccine recipients as early as day 7 post transplant. In comparison a gB antibody response was only detectable much later (>35 days) in the placebo group. One possible interpretation is that vaccinated individuals were primed to generate a rapid response from memory when they encountered HCMV from the donor organ. However, it was important to note that it was not simply the magnitude of the gB response that was important post-transplant. This is in contrast to pre-transplant where the gB antibody titre in response to vaccination was a clear correlate of protection against duration of viraemia The rapid onset of IgG gB and a general lack of IgM responses against gB  is consistent with boosting of a pre-existing response in the primed B cells of these T-cell suppressed hosts. Indeed, the comparative delay seen in the placebo group would argue that immune-suppressed patients cannot make novel antibody responses against HCMV during the early stages post-transplant . Here it is important to re-iterate that no statistical correlation between total antibody levels post-transplant and outcome could be made. For example, some vaccinated individuals had evidence of less boosting post-transplant but were protected. The possibility that pre-formed antibodies may have prevented transmission of virus from the donor (which would prevent downstream viraemia and a boosting effect) should be examined in future vaccine studies with larger patient cohorts than available here.Perhaps the most important aspect of this study is the suggestion that vaccine can induce a neutralizing antibody response against gB but that this was only revealed post-transplant. Evidence of neutralizing activity was observed as early as 7 days post-transplant in some vaccinees, with the majority of them having antibodies that were able to neutralize infection at day 35 post-transplant. An important aspect of this was that the neutralizing antibody response in the vaccinees was negated using the gB antigen. In contrast, the gB antigen did not reverse the neutralizing activity of sera from healthy seropositive donors. Essentially, the vaccinees have only generated antibody responses against gB and thus the neutralizing response is dominated by gB responses at those early times post transplant. However, it is likely that if these individuals seroconvert the dominance of gB antibodies in the repertoire will be reduced. The gB antibody phenotype of the response seen in our vaccinees at 7–35 days post transplant rarely happened in the recipients of placebo although, over time, they did also develop neutralizing antibody responses. The development of these responses in our placebo controls were possibly linked with a reduction of immune-suppression over time and probably included responses targeted against multiple antigens on HCMV. However, in the context of vaccination, despite a very limited sample size, the analyses suggest that neutralization may be inversely correlated with the duration of viraemia- a possibility that should be examined in future larger clinical trials.This in itself raises some important questions. The role of neutralizing antibodies for the activity of the gB vaccine has been debated. In two separate analyses, vaccination did not induce measurable levels of neutralizing antibody responses against gB ,17. HoweTaken together, despite the severe limitations of sample size that have limited many of the follow-up analyses presented here, the data suggest that vaccine recipients were immunologically primed by vaccination and that transplant, and thus challenge with HCMV, induced functional antibody responses that had been primed by vaccination. A correlate of this was that these vaccine recipients inhibited viral infection more effectively in comparison to the placebo group. We suggest that future studies on HCMV vaccines in SOT should include analysis of the sera (and lymphocytes) collected post transplantation to allow comprehensive follow up analyses to further define the mechanistic basis for the protective effect seen with the gB/MF59 vaccine and to begin to assess the impact of viral infection on the immune response generated by vaccination against HCMV in seronegative individuals.S.P. and F.D.P. are employees of Sanofi Pasteur. All other authors declare no conflicts of interest.


Dengue fever is one of those unique diseases where host immune responses largely determine the pathogenesis and its severity. Earlier studies have established the fact that dengue virus (DENV) infection causes haemorrhagic fever and shock syndrome, but it is not directly responsible for exhibiting these clinical symptoms. It is noteworthy that clinically, vascular leakage syndrome does not develop for several days after infection despite a robust innate immune response that elicits the production of proinflammatory and proangiogenic cytokines. The onset of hyperpermeability in severe cases of dengue disease takes place around the time of defervescence and after clearance of viraemia. Extracellular vesicles are known to carry biological information  from their cells of origin and have emerged as a significant vehicle for horizontal transfer of stress signals. In dengue virus infection, the relevance of exosomes can be instrumental since the majority of the immune responses in severe dengue involve heavy secretion and circulation of pro-inflammatory cytokines and chemokines. Here, we present an updated review which will address the unique and puzzling features of hyperpermeability associated with DENV infection with a special focus on the role of secreted extracellular vesicles. Aedes aegypti and to a lesser extent by A. albopictus. Primary dengue infection and disease is manifested as mild and self-limiting illness in the majority of individuals. Dengue Haemorrhagic Fever (DHF), popularly known as “severe dengue,” was first recognized in the Philippines and Thailand during dengue epidemics. Severe dengue has now spread in most Asian and Latin American countries and poses a heavy burden on healthcare cost, hospitalization and death in both children and adults (WHO). Recent WHO reports indicate 390 million dengue infections per year although actual numbers of dengue cases are often underreported and misclassified due to the similarity of symptoms with other viral diseases; still almost 3.9 billion people, spanning around 128 countries, are at risk of dengue infection [Dengue fever is caused by the infection of dengue virus. It is a mosquito-born disease identified just 100 years ago [Flavivirus genus contains more than 70 viruses which are transmitted by arthropods [Dengue virus belongs to family flaviviridae. The thropods . To datethropods . DENV-5 Patients with dengue infection progress through three typical clinical phases; the febrile phase, the critical phase and the recovery phase . It begins with an initial incubation period of typically 3–7 days followed by a sudden onset of high fever along with high viraemia, known as the febrile phase . Some patients proceed to the critical phase which lasts for 24–48 h and is associated with plasma leakage. Interestingly some patients might revert to the recovery phase without experiencing plasma leakage . In a feet.al. [Antibody-Dependent Enhancement) and it was first reported in 1970 by Halstead [Vascular hyperpermeability and plasma leakage are two major hallmarks of DHF. A plethora of research carried out around the world has attempted to pinpoint the cause of this severe dengue. The roles of many cytokine mediators, mast cell products , inflammet.al. . Since tet.al. . This phHalstead .Here we will provide a detailed explanation for DHF and address all currently prevalent hypotheses of DHF and try to highlight their major limitations in explaining the complexities of the disease..ADE is a very unique process, where pre-existing virus-specific antibodies help more viral entry in host cells and thereby help the virus rather than host. Other than DENV, HIV-1, Ebola, and Influenza viruses are also known to exhibit ADE. These viruses share a few common attributes such as preferential replication in macrophages/monocytes, ability of persistence and a greater antigenic diversity .The idea that antibodies produced during a primary DENV infection might be a prime cause of severe DENV condition (through ADE) remained controversial for a long time. However, this conclusion was based on extensive epidemiological studies. Severe dengue disease is frequently observed among first-time-infected infants born to DENV-immune mothers. Those children who had developed a mild or asymptomatic dengue previously were found highly susceptible for developing haemorrhagic fever or severe dengue when infected by a different DENV serotype , 23.in vitro [Many recent reports have kept this debate alive whether ADE alone is enough to explain dengue haemorrhagic fever. First, many reports of dengue haemorrhagic fever in the primary infection  indicate that antibody-dependent enhancement is not an essential pre-requisite for plasma leakage . Second,in vitro . Thus, Ain vitro, at least in the early phase of infection [Dengue virus is neither a direct causative agent for hyperpermeability nor the acute cytokine storm could be the prime reason for hyperpermeability nfection . In addinfection , 27. It nfection . In contnfection , 29.These varying observations indicate towards the involvement of a much more complex series of events related to hyperpermeability and suggest a lack of direct cause and effect relationship. Extremely low percentage of dengue virus-infected cells in blood and yet massive vascular leakage syndrome across the host suggests the role of some bystander/passive effect for causing this increased vascular permeability. The source/cause of such hyperpermeability could be diffusible and capable of intercellular communication without coming in direct contact with viral particle or antigens. This would also give virus a survival advantage since they would be able to escape the host immunity but still transmitting their characteristic vascular pathology in the host.Original antigenic sin is also known as Hoskin’s effect. This phenomenon can be understood as “preferential boosting of pre-existing cross-reactive antibodies upon sequential infections” . This ma+ and CD8+ T cells are reported to help resolve DENV infection. Upon primary DENV infection, serotype-specific CD4+ and CD8+ T-cell responses have been observed [+ T cells have been discovered predominantly against NS3, NS1, and envelope antigenic epitopes. CD4+ T cells specific for NS3 proliferate and usually produce IFN-γ and lyse the virus-infected cells [+ T cells produce a higher ratio of TNF-α to IFN-γ upon stimulation with a heterologous antigen as compared to stimulation with homologous serotype [in vivo than patients suffering from only DF (based on serum markers of activation) [In the case of DENV infection, cell-mediated immunity; both CD4observed . DENV-sped cells . Howevered cells . Serotyped cells . The preserotype , 38. It serotype . It is aserotype . Earlierivation) , 42–44. ivation) . It also“Original antigenic sin” in DENV infections also has a protective role in addition to its role in pathogenesis . In a rein vitro by DENV with no significant apoptosis [Studies report only a small percentage (2%) of endothelial cells to be productively infected poptosis . This prpoptosis . This stpoptosis , it is lIn general, EVs have been demonstrated to exert a bidirectional regulatory effect on host–pathogen interaction. It can deliver its content to either promote the infection or suppress the infection. Pathogenic interests are promoted via (1) transmission of pathogen-related molecules like viral RNA or proteins, (2) immune escape of pathogen, and (3) dampening of the host immune responses. On the other hand, EVs are shown to negatively regulate pathogen via transmission and induction of antiviral responses in host immune cells  . CurrentAll kind of immune cells, like DCs, T cells, B cells, mast cells, monocytes, and macrophages, are engaged at some time point in dengue virus life cycle and cross-talk between them seems to happen in a very dynamic and temporal fashion. Exosomes released by these cells suggest the participation of these microparticles as a preferred choice of transportation. Exosomes are even shown to contribute towards an optimal T-cell response rather than direct antigen presentation by macrophages and DCs . Like otHere we are going to review all currently available reports on the role of EVs and discuss how they can either contribute positively or negatively for DENV pathogenesis . Figure Virus of Alternative Morphology (VAM) [In a mosquito gut cell line C6/36 which is very frequently used for DENV replication, DENV2/DENV3-infected cells were reported to secrete EVs . These Egy (VAM) . Since egy (VAM) .Dengue virus (DENV) activates platelets via CLEC2 (C-type lectin like receptor 2) to release EVs which includes both exosomes and microvesicles . DENV-inExosomes have been shown to deliver IFITM3 (Interferon-induced transmembrane protein 3) from DENV-infected cells to non-infected cells. Exosomes carrying IFITM3 transmit antiviral activities from cell to cell during DENV infection . IFITMs Interestingly, another study revealed that IFITM proteins could interfere with the antibody-dependent enhancement (ADE) phenomenon during the secondary DENV infection, which bypassed the IFN-mediated restriction . Chan etVarious microarray analyses demonstrated aberrant miRNA expression in patient’s plasma as a hallmark of DENV infection. Total plasma samples, endothelial cells, macrophages and dendritic cells, all of them have been reported to display altered microRNA profiling upon DENV infection . EndotheApart from endothelial permeability, DENV disease severity is also expressed in the form of hyperactivation or unregulated expression of pro-inflammatory gene networks, a phenomenon is also known as “Cytokine Storm.” Here again microRNAs play a very important role in regulating almost all immune regulatory pathways in various immune cells. miR-146, miR-155, let-7a, miR-4279,miR-30, miR-451, miR-106b, and others have been found to be directly regulating cytokine storm via regulating IL-6/8, CXCL9/10/11, CCL5, TNF-a, IL-10, etc. . This fiSeveral examples for innate immunity modulation by exosomal microRNA are reported so far , 79. SpeA list of exosomal microRNAs targeting endothelial junctional protein, thereby becoming potential contributor for vascular disintegration, is given in Earlier reports showed that exosomes carry lesser amount of small RNA than cellular levels, but interestingly those small RNA fraction were enriched in miRNAs . Anotheret.al. demonstrated that the level of microRNAs loaded in exosomes is determined by the relative levels of the microRNA and their target mRNA inside cell. This is likely to happen if there is an abundance of the target mRNA of certain microRNA, in that case the microRNA will stay bound with target mRNA and this will reduce the loading of microRNA in the exosomes. In the case of higher levels of the microRNA relative to its target mRNA, enhanced loading of the microRNA will be observed in exosomes. This interesting mechanism also suggested that exosomal release pathway is not just to secrete viral factors but also to counter balance the microRNA:mRNA homeostasis in the cells[A recent study demonstrated that microRNAs released in exosomes reflect the cellular microRNA expression profile , 83. It the cells.The most staggering aspect of exosomes in DENV pathogenesis is that exosomes or microvesicle-mediated communication can allow the virus to respond or modulate the cellular microenvironment even in the absence of active viral replication. In this review, we have attempted to highlight the notion that a single theory of DHF cannot explain the whole scenario alone. DHF is a phenomenon which occurs at multiple places in the host despite the fact that DENV is not homogenously distributed either temporally or spatially. DENV-infected cells secrete microvesicles loaded with a variety of molecular cargo. Further study of virally modified microvesicles/exosomes will eventually resolve their roles in DENV pathogenesis. An obvious functional overlap between exosome biogenesis and viral budding encourages us to hypothesize that exosomes should be a key influencer for virus life cycle. Exosome research and their precise role in DENV pathogenesis and other viruses are still in infancy. A multidimensional understanding of exosome biology and its significance in viral pathogenesis would certainly give us many useful windows to interfere with viral replication and Pathogenesis.


Non-ST elevation acute coronary syndromes (NSTE-ACS) may arise from moderately stenosed atherosclerotic lesions that suddenly undergo transformation to vulnerable plaques complicated by rupture and thrombosis.  Assessment and tissue characterization of the coronary atherosclerotic lesions among NSTE-ACS patients compared to those with stable angina.  Evaluation of IVUS studies of 312 coronary lesions was done by 2 different experienced IVUS readers, 216 lesions in 66 patients with NSTE-ACS (group I) versus 96 lesions in 50 patients with stable angina (group II). Characterization of coronary plaques structure was done using colored-coded iMap technique. p=0.001). Body mass index (BMI) was significantly higher in group II while triglycerides levels were higher in group I . History of previous MI and PCI was significantly higher in group I . The coronary lesions of NSTE-ACS patients had less vessel area , stenosis percentage , and plaque burden  with negative remodeling index  compared to the stable angina group. On the other hand, they had more lipid content , necrotic core , and calcifications  while less fibrosis  compared to the stable angina patients. Syntax score and core composition especially calcification and lipid content were significant predictors to NSTE-ACS.  The Syntax score was significantly higher in group I compared to group II (18.7 ± 7.8 vs. 8.07 ± 2.5,  The vulnerability rather than the stenotic severity is the most important factor that predisposes to non-ST segment elevation acute coronary syndromes. The vulnerability is related to the lesion characteristics especially lipidic core and calcification while lesion fibrosis favours lesion stability. Acute coronary syndromes frequently arise from erosion of vulnerable plaques and subsequent thrombosis , 2 whilePrevious studies have noted that histologic thumbprints of these vulnerable plaques were characterized by thin-capped atheroma with a lipid-rich core, few smooth muscle cells, numerous macrophages, adventitial inflammation, and positive remodeling –6.The main challenge faced was that ACS often arises from lesions with only mild to moderate stenosis. Accordingly, disclosure of potentially vulnerable plaques may promote prevention of cardiovascular events , 8.Our objective was to study the coronary atherosclerotic lesion characteristics that predict the occurrence of non-ST segment elevation acute coronary syndromes compared to those with stable angina.We did this cross-sectional study on 116 patients (50 patients with stable angina and 66 patients with non-ST segment elevation acute coronary syndromes) subjected to coronary catheterization between the periods from January 2017 to January 2018.Stable angina is defined as a clinical syndrome characterized by discomfort in the chest, jaw, shoulder, back, or arm. It is typically aggravated by exertion or emotional stress and relieved by nitroglycerin with no change in character for sixty days. Angina usually occurs in patients with CAD involving ≥1 large epicardial artery .Chest pain in absence of persistent ST elevation with elevated cardiac biomarkers of necrosis is suggestive of NSTE-ACS .Informed consents were obtained from all individual participants included in this study. The study had been approved by the institutional research ethical committee.Patients with acute ST elevation myocardial infarction (STEMI), previous coronary artery bypass grafting (CABG), renal impairment, and thrombocytopenia were excluded.Coronary angiography and interventions were done using Philips  and Siemens (Axiom Artis DFC 35875) with 15 frames per second (fps) imaging speed.Three hundred twelve coronary lesions were assessed by IVUS during diagnostic angiography, 216 lesions in 66 patients with NSTE-ACS (group I) versus 96 lesions in 50 patients with stable angina (group II).We used iLab™ Ultrasound Imaging System  to get IVUS runs, after administration of 200 mcg intracoronary nitroglycerin, using a 40 MHz 6F compatible catheter (Atlantis SR Pro).Image acquisition was done through retrograde-automated transducer pullback at 0.5 mm/second. The pullback started from at least 10 mm distal to the studied lesions as the distal reference segment was defined as the site with the largest lumen distal to a stenosis but within the same segment .Based on images depicted during pullback of the transducer, the lesion was defined as the image slice with the smallest lumen cross-sectional area .The measurements were taken according to the American College of Cardiology guidelines, and reporting was done by two experienced IVUS readers .Lumenal and external elastic membrane (EEM) cross-sectional areas (CSAs) were measured for each 1 mm of axial length, and then, plaque plus media (P&M) CSA was calculated by EEM CSA minus lumen CSA.Plaque burden was calculated by P&M CSA divided by EEM CSA. The lesion was considered significant when percent area stenosis >70%.The distal and proximal vessel reference segments were the most apparently normal segments within 10 mm distal and proximal to the coronary lesion.The remodeling index (RI) was calculated by EEM CSA divided by the mean reference CSA .The colored-coded iMap technique was used to get the characteristics of coronary plaques structure.Precoded data were entered on the computer using “Microsoft Office Excel Software” program (2010) for Windows. Data were then transferred to the Statistical Package of Social Science Software program, version 21 (SPSS), to be statistically analyzed.Data were summarized using mean, standard deviation, median, and interquartile range for quantitative variables and frequency and percentage for qualitative ones.t-test or one-way ANOVA with Tukey's post hoc test for quantitative variables and the chi-squared test or Fisher's exact test for qualitative ones.Comparison between groups was performed using the independent sample Univariate regression analysis had been used to determine potential predictors of ACS.P values less than 0.05 were considered statistically significant and less than 0.01 were considered highly significant. Graphs were used to illustrate some information.Body mass index (BMI) was significantly higher in the stable angina (SA) group while the triglyceride level was higher in the NSTE-ACS group.History of previous MI and PCI was significantly higher in the NSTE-ACS group .The study involved 312 coronary lesions, and the culprit lesions were 134 for which stenting was done. LM lesions were higher in the NSTE-ACS patients compared to SA patients. The Syntax score was significantly higher in the NSTE-ACS group compared to the SA group .Notably, the coronary lesions of NSTE-ACS patients had significantly less vessel area with negative remodeling index compared to those with the SA group. Also, the plaque burden and percent area stenosis were significantly smaller than those of the SA group .Regarding lesion characteristics, NSTE-ACS group lesions had significantly more lipidic content, necrotic core, and calcifications with less fibrosis compared to the stable angina patients  and 2.Univariate regression analysis had been used to determine potential predictors of NSTE-ACS and had showed that the Syntax score and core composition especially calcification and lipid composition were significant predictors .Plaque destabilization is a biomechanical phenomenon depending on complex interactions between applied shear stresses, resulting in reactive oxygen species production and inflammation, blood laminar or turbulent flow characteristics, coronary lesion structural features, and biological processes that determine mechanical strength , 13–17.Plaque rupture refers to transmural fissuring of the atheroma fibrous cap and exposure of the underlying necrotic core with its proinflammatory and thrombogenic activities to circulating blood. It is the commonest form of plaque destabilization. The ruptured plaque usually has the features of a thin cap fibroatheroma , 18–20; Our study used IVUS to assess the atherosclerotic burden and the plaque vulnerability in culprit and nonculprit vessels during cardiac catheterization of patients with stable angina and NSTE-ACS. Both groups had similar risk factors of atherosclerosis except for hypertriglyceridaemia and BMI. NSTE-ACS patients had moderately stenosed coronary lesions with less plaque burden but negative remodeling index as compared to patients with stable angina.Our results were similar to those of Ghaffari et al.  which coOur study showed that NSTE-ACS patients had a higher ratio of lipidic content, dense deep calcium, and necrotic core components, while less fibrotic content compared to stable angina patients. This could explain the occurrence of ACS despite having moderately stenosed coronary lesions with less plaque burden. The lesion characteristics were similar to those in Nakamura et al.'s study  which coThe PROSPECT  study  confirmeOur study revealed that NSTE-ACS patients had lesions with significantly negative remodeling index as compared to patients with stable angina, which was consistent with the results of Koo et al.  and FernOur results revealed that SYNTAX score and core composition, especially calcification and lipid composition, were significant predictors for NSTE-ACS. This was different to a study by Zheng et al. , in whicThe plaque vulnerability hypothesis was used to better describe the unpredictability of the future course of atherosclerosis. Vulnerable plaques have been defined as those plaques prone to becoming culprit plaques causing acute coronary events, regardless of stenosis, shape, or destabilization, without taking into account the effect of exogenous factors, such as shear stresses, blood laminar or turbulent flow characteristics, and vascular anatomy and function  .Finally, coronary lesions, even with less plaque burden, should not be underestimated because they could be vulnerable plaques and predispose to future cardiac events. IVUS helps to detect plaques morphology and vulnerability, but the PREDICTION study  only demThe clinical applicability of vulnerable plaques concept has led to advances in our understanding of pathogenesis and management of atherosclerosis. Patients with vulnerable nonculprit lesions require systemic approach rather than localized treatment. Intensifying medical treatment and life-style change may reduce the atherosclerosis burden and future cardiac events.The current study was a single-center study with a small number of patients included.The vulnerability rather than the stenotic severity is the most important factor that predispose to Non ST segment elevation acute coronary syndromes. The vulnerability is related to the lesion characteristics especially the lipidic core and calcification while lesion fibrosis favors lesion stability.


PRSweb that provides detailed PheWAS results and allows users to directly compare different PRS construction methods.Polygenic risk scores (PRS) are designed to serve as single summary measures that are easy to construct, condensing information from a large number of genetic variants associated with a disease. They have been used for stratification and prediction of disease risk. The primary focus of this paper is to demonstrate how we can combine PRS and electronic health records data to better understand the shared and unique genetic architecture and etiology of disease subtypes that may be both related and heterogeneous. PRS construction strategies often depend on the purpose of the study, the available data/summary estimates, and the underlying genetic architecture of a disease. We consider several choices for constructing a PRS using data obtained from various publicly-available sources including the UK Biobank and evaluate their abilities to predict not just the primary phenotype but also secondary phenotypes derived from electronic health records (EHR). This study was conducted using data from 30,702 unrelated, genotyped patients of recent European descent from the Michigan Genomics Initiative (MGI), a longitudinal biorepository effort within Michigan Medicine. We examine the three most common skin cancer subtypes in the USA: basal cell carcinoma, cutaneous squamous cell carcinoma, and melanoma. Using these PRS for various skin cancer subtypes, we conduct a phenome-wide association study (PheWAS) within the MGI data to evaluate PRS associations with secondary traits. PheWAS results are then replicated using population-based UK Biobank data and compared across various PRS construction methods. We develop an accompanying visual catalog called  In the study of genetically complex diseases, polygenic risk scores (PRS) synthesize information from multiple genetic risk factors to provide insight into a patient’s inherited risk of developing a disease based on his/her genetic profile. These risk scores can be explored in conjunction with health and disease information available in electronic medical records. PRS may be associated with diseases that may be related to or precursors of the underlying disease of interest. In this paper, we demonstrate how PRS can be used in concert with the medical phenome to better understand the etiology of disease subtypes nested within a broad disease classification. This is done by examining the shared and distinct genetic risk factors across the related but heterogeneous disease subtypes and also through our comparison of the secondary associations across the phenome corresponding to the subtype specific PRS. We consider several PRS construction methods in our study. This framework of analysis is enabled by access to electronic health records and genetics data. Leveraging and harnessing the rich data resources of the Michigan Genomics Initiative, a biorepository effort at Michigan Medicine, and the large population-based UK Biobank study, we investigated the primary and secondary disease associations with PRS constructed for the three most common types of skin cancer: melanoma, basal cell carcinoma and cutaneous squamous cell carcinoma. The underlying risk factors of genetically complex diseases are numerous. Genome-wide association studies (GWAS) on thousands of diseases and traits have made great strides in uncovering a vast array of genetic variants that contribute to genetic predispositions to disease . In ordeG1, G2, ⋯GK, the depth of the list or the number of markers (K), and the choice of the weights In the post-GWAS era and with the availability of large biobank data from multiple sources, there is great interest in using gene-based biomarkers such as PRS for risk stratification and exploration of disease etiology. However, it is not always clear how best to construct a PRS for a particular phenotype. A PRS of the general form  results , 9. Whil results , 10, it In this paper, we first explore strategies for constructing a PRS using markers and weights obtained from either the latest GWAS or the NHGRI-EBI GWAS catalog that have reached genome-wide significance. We compare the PRS in terms of their performance  for the For the chosen PRS corresponding to each skin cancer subtype, we perform a phenome-wide association study (PheWAS) relating the PRS to the EHR-based phenome of MGI. We call such a study a PRS-PheWAS . PRS-Pheunique to that subtype’s chosen PRS. We further construct a composite PRS for general skin cancer consisting of loci common among all subtypes’ PRS. While merging distinct clinical entities into a compound PRS may seem counterintuitive in terms of specificity, such an approach may increase power to identify dermatological features through PheWAS that are shared by all three subtypes. Such features may help provide insight into the shared biological etiology and disease development across disease subtypes.We then describe an approach for using PRS to more directly evaluate the shared and unique genetic architecture of disease subtypes and identify shared and unique secondary phenotype associations related to this genetic architecture. We define a new PRS for each skin cancer subtype using loci -8). However, it is likely that there are additional risk variants below this threshold that could be associated with the trait but have not reached statistical significance  /OCA2 [MIM 611409], IRF4 [MIM 601900], MC1R [MIM 155555], RALY [MIM 614663], SLC45A2 [MIM 606202] and TYR [MIM 606933]) were previously found to be associated not only with skin cancer traits, but also with pigmentation traits of skin, eyes and hair .Fig B inTable J inThe ICD-code-based collection of UK Biobank GWAS results did not include basal cell carcinoma or squamous cell carcinoma subtypes; rather, it included only the merged trait ‘non-epithelial cancer of skin’ Fig B in. Thus, wTable D in-4. For this p-value cutoff, the melanoma PRS had similar AUC and OR for the melanoma and basal cell carcinoma phenotypes. This p-value cutoff represents the least conservative inclusion cutoff with 1,193 included loci, and its results indicated that inclusion of too many suggestive SNPs at lower thresholds may reduce PRS performance. However, we also note that the most conservative cutoff (5x10-9) produced a PRS that was based on only six loci, which had a weaker OR and AUC compared to other PRS created with less stringent cutoffs. The best performance in terms of AUC and OR relating to the melanoma phenotype were observed for p-value thresholds 5x10-7 and 5x10-8, which included 13 and 9 loci respectively. The small number of loci identified by this method at more conservative p-value cutoffs may be driven by the lower sample size for melanoma in the UK Biobank compared to the published melanoma GWAS meta-analyses . We note that the melanoma PRS constructed using the UK Biobank summary statistics produced lower AUC across all p-value thresholds than was seen for the latest GWAS and GWAS catalog PRS construction methods.-4), for which the AUC and odds ratio were smaller. Overall, the highest AUCs and strongest OR signals for both PRS and all skin cancer phenotypes were found at depths of 5x10-7 and 5x10-8.Among the skin cancer subtypes, the PRS constructed for overall skin cancer was most strongly associated with basal cell carcinoma across all p-value thresholds, with odds ratios ranging from 1.4  to 1.64 . Among the PRS, the overall skin cancer PRS had the greatest discrimination for the overall skin cancer phenotype. Overall skin cancer and melanoma PRS had similar performance in terms of discrimination for the melanoma phenotype across various depths. The overall skin cancer PRS tended to be more strongly associated with and have similar or slightly better discrimination for the overall skin cancer phenotype compared to the melanoma PRS, indicating that the overall skin cancer PRS was more accurate at predicting the overall skin cancer phenotype than the melanoma PRS. The overall skin cancer PRS had very similar association with and discrimination abilities for the overall skin cancer phenotype across all p-value thresholds except the least conservative  and Fig K (melanoma) in-7 and 5x10-8 produced very similar results, and other depths tended to identify fewer phenotypes associated with the corresponding PRS. Phenotypes that were associated with the PRS at other depths had weaker associations. PRS-PheWAS using the melanoma PRS and the overall skin cancer PRS produced somewhat different results. For example, the melanoma PRS at different depths did not identify strong associations with “diseases of sebaceous glands”, which is similar to previous PRS-PheWAS results for mPRS in MGI and UKB. In contrast, the overall skin cancer PRS did identify associations with “diseases of sebaceous glands” or its subcategories for all depths except 5x10-5 and 5x10-4. Fig L inIn addition to associations with the primary and related phenotypes, we explored associations between PRS constructed at various UK Biobank summary statistic depths and secondary phenotypes. Table E in-8 depth in terms of associations with the primary phenotype. Using the PRS with a percentage of assumed causal variants producing the best pseudo R2 statistic from Table E inFigure M inWe evaluated the performance of PRS constructed using the LDpred algorithm, which incorporates millions of SNPs into the PRS definition. PRSweb available at https://statgen.github.io/PRSweb that enables interactive exploration of the PheWAS results for each of the skin cancer subtypes and different PRS construction methods explored in this paper, for both the MGI and UK Biobank phenomes. PRSweb shows PRS-PheWAS plots with various choices of PRS in the drop-down menu  have good performance in terms of specificity for the primary phenotype. All PRS were positively associated with all skin cancer phenotypes under consideration. This suggests that each individual PRS may capture some shared genetic contributions for disease development across skin cancer subtypes.Appendix 1 inFor each skin cancer subtype, we performed a PRS-PheWAS to identify secondary phenotypes that are associated with the corresponding PRS. We identified many dermatological features in addition to the primary phenotype, indicating the ability of PRS to reproduce associations with the primary phenotype even after multiple testing corrections and covariate adjustment. The majority of these associations were replicated in a PRS-PheWAS performed for the UK Biobank phenome. Our analyses identified actinic keratosis, which is believed to be a precursor to squamous cell and basal cell carcinoma, as an independent predictor of basal cell and squamous cell carcinoma, and we demonstrated that incorporating the PRS in addition to clinical information improved discrimination for future skin cancer diagnoses  were also loci that had been associated with human pigmentation traits and/or harbor key genes of the biochemical pathway of melanogenesis [In an additional analysis, we identified loci that were shared among all three skin cancer subtypes’ PRS. Loci overlap between the PRS for the three subtypes may indicate factors related to common biology between the subtypes. We noted that all shared loci  GWAS may provide a better source of evidence to use for PRS construction due to larger sample sizes and  more carefully curated data. The latest GWAS and GWAS catalog methods produced PRS with similar performance in terms of their associations with and discrimination for the primary phenotype used to construct the PRS for both basal cell carcinoma and melanoma. The latest GWAS method produced better results for squamous cell carcinoma.-8 or 5x10-7 resulted in the best performance, but further increasing the p-value threshold resulted in reduced performance. Crucially, we also observed stronger associations between the PRS and secondary phenotypes for PRS constructed using depths of 5x10-8 and 5x10-7. These results suggest that some benefit may be observed by incorporating loci that do not reach significance into the PRS construction but incorporating too many loci with larger p-values may not improve the predictive ability of the PRS (for both primary and secondary phenotypes). However, this gain or reduction in PRS performance may depend on the phenotype of interest and on the prevalence of the phenotype in the analytical sample.In the second PRS construction strategy, we use UK Biobank summary statistics at different p-value depths to construct PRS. We found that incorporating additional loci that did not reach genome-wide significance did improve the PRS’ ability to discriminate cases from controls for the primary phenotype up to a point. In particular, PRS constructed using SNPs with p-values less than 5x10In the third PRS construction strategy, we use the LDpred method to construct PRS using the whole spectrum of observed genetic information. For melanoma, the LDpred PRS which modelled smaller fractions of causal variants were favored over the ones modelling larger fractions. While the PRS construction with LDpred performed similarly to the various depth approach in our particular study, its computational cost was substantially higher. However, recent work indicated that LDpred might outperform pruning and thresholding approaches when larger training data sets are available .In our study, PRS associations with the primary phenotype were generally stronger using the latest GWAS and GWAS catalog-based PRS than for the PRS constructed using LDpred or using UK Biobank summary statistics at different depths. Since the underlying case numbers in the discovery GWAS for the former were substantially larger than the case numbers in the UK Biobank GWAS, no direct comparison between approaches was possible, and also because the required full summary statistics of the contributing and larger skin cancer case-control studies of the latest GWAS and GWAS catalog entries were not made available. Additionally, these simpler PRS construction strategies appeared to more clearly differentiate between related subtypes than the genome-wide PRS construction methods. All three PRS construction strategies produced many similar secondary associations from PRS-PheWAS, as shown in PRSweb (see Web resources) that provides PRS-PheWAS results for the various skin cancer phenotypes for PRS constructed using the different methods explored in this paper. PRSweb will provide a routine way to compare different PRS construction methods and to explore PRS-PheWAS results in detail. Additionally, PRSweb provides the PRS construction details, which researchers can download and use in their own analyses. In the future, we plan to extend this online platform to include PheWAS for many other cancer phenotypes, which will make this online platform a general tool for identifying phenotypes related to particular types of cancer.As a product of this study, we provide an online visual catalog Fig N inOne limitation of the generalizability of this study comes from the homogeneous race profile of MGI and UK Biobank. UK Biobank consists of subjects of primarily European descent, and we restricted our analyses to subjects of European descent in MGI (excluding about 10% of the subjects in MGI) in order to ensure greater comparability between the two datasets. Additionally, many of the existing GWAS were conducted on European populations, and we wanted to consider similar samples when comparing the performance of PRS constructed using summary statistics from European populations. Unlike UK Biobank, MGI is not a population-based sample; rather, it is a sample of patients recruited from a large academic medical center. Patients were recruited prior to surgery through the anesthesiology department, and therefore they may present a potential for selection bias. PRS-PheWAS results are susceptible to collider bias caused by PRS relationships with both skin cancer diagnosis and other diseases related to sampling. The exclusion PheWAS strategy attempts to overcome this obstacle by evaluating PRS associations with secondary phenotypes only in the subjects without a skin cancer diagnosis. This approach does not remove the possibility of sampling bias, but it may help reduce the impact of sampling on the estimated PRS-phenotype associations. The chosen design of matched case-control studies can reduce bias and detection of false positives compared to the unmatched analysis, but it is typically less powerful than the unmatched analysis Fig N in.S1 Section 1, we discuss this issue in more detail, and we provide a sensitivity analysis exploring the impact of misclassification on study results in Fig O inAn additional limitation for all EHR-based phenome-wide studies is the potential for bias due to phenotype misclassification. In https://statgen.github.io/PRSweb University of Michigan Medical School Central Biorepository; https://research.medicine.umich.edu/our-units/central-biorepository UK Biobank; http://www.ukbiobank.ac.uk/ UK Biobank GWAS summary statistics; https://tinyurl.com/UKB-SAIGE TOPMed variant browser, https://bravo.sph.umich.edu/freeze5/hg38/ TOPMed program, https://www.nhlbi.nih.gov/science/trans-omics-precision-medicine-topmed-program Minimac4; https://genome.sph.umich.edu/wiki/Minimac4 BCFtools; https://samtools.github.io/bcftools/bcftools.html KING; http://people.virginia.edu/~wc9c/KING/ FASTINDEP; https://github.com/endrebak/fastindep PLINK; https://www.cog-genomics.org/plink2/ Eagle; https://data.broadinstitute.org/alkesgroup/Eagle/ UCSC Genome Browser; http://genome.ucsc.edu/ R; https://cran.r-project.org/ NHGRI-EBI GWAS Catalog; https://www.ebi.ac.uk/gwas/ dbSNP; https://www.ncbi.nlm.nih.gov/projects/SNP/ Imputation server; https://imputationserver.sph.umich.edu/ Jinja, https://github.com/pallets/jinja Locuszoom, https://github.com/statgen/locuszoom.PRSweb; S1 TextThis file contains supporting Appendices 1–2, Figures A-O and Tables A-E.(PDF)Click here for additional data file.S1 FileThis Excel file contains the following tables: Sheet 1: Table F, ICD9 codes to PheWAS code mapping tables for skin cancer; Sheet 2: Table G, ICD10 codes to PheWAS code mapping tables for skin cancer; Sheet 3: Table H, MGI and UK Biobank phenome summary; Sheet 4: Table I, GWAS Catalog and latest GWAS risk SNP selection; Sheet 5: Table J, UKB GWAS risk SNP selection; Sheet 6: Table K, Omnibus table of significant results.(XLSX)Click here for additional data file.


Prosthetic valve endocarditis (PVE) is a life-threatening systemic infection involving a high mortality rate and severe complications, including perivalvular abscess. Early diagnosis and detection of PVE continue to be challenging in clinical settings.18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) was performed; revealing intense hyper-metabolism above the aortic valve prosthesis with a greater intensity at the posterior end, confirming a diagnosis of aortic PVE complicated with perivalvular abscess.A 64-year-old man with a history of mechanical aortic valve implantation 12 years prior was referred to our hospital with a major complaint of high fever and was admitted. Although results of three blood culture tests at admission were negative, transthoracic echocardiography, and transoesophageal echocardiography (TOE) were performed to exclude the possibility of PVE; both, however, were inconclusive. Subsequently, 18F-FDG PET/CT can play a central role in diagnosing PVE. However, this new imaging modality often fails to differentiate thrombi, soft atherosclerotic plaques, or foreign body reactions on the surface of prosthetic valves. In this report, we have successfully enhanced the diagnostic accuracy of 18F-FDG PET/CT by focusing on perivalvular involvement, which could be a key finding, because intense 18F-FDG uptake surrounding the aortic annulus was consistent with the thickened area within the aortic annular region observed in the TOE examinations.Considering the intermediate suspicion of PVE despite negative TOE and negative blood culture tests,  Prosthetic valve endocarditis (PVE) can often be a major diagnostic challenge owing to the difficulties in the interpretation of conventional echocardiography, particularly in patients with low-to-intermediate probability for PVE based on the modified Duke criteria.18F-fluorodeoxyglucose positron emission tomography and computed tomography angiography aided in the diagnosis of PVE and its complications, shedding light on the additional roles of the new imaging modalities.We describe a case of PVE complicated by perivalvular abscess where the combination of Definitive diagnosis of prosthetic valve endocarditis (PVE) is clinically challenging because of its varying clinical presentation, microorganisms involved and patient profile. Prosthetic valve endocarditis entails high initial mortality and morbidity rates with a high risk of complications, including perivalvular abscess and fistulae during follow-up and often requires surgical intervention.18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) has been advocated as an alternative diagnostic imaging modality for PVE, particularly when echocardiography and blood culture results are inconclusive in reaching a definitive PVE diagnosis.18F-FDG PET/CT can detect distant emboli, metastatic infections and perivalvular involvement along with coronary anatomy when combined with CT coronary angiography.Recently, 18F-FDG PET/CT and CT coronary angiography revealed intense hyper-metabolism above the aortic valve prosthesis with greater intensity at the posterior end and absence of obstructive coronary artery, thereby confirming the diagnosis of aortic PVE complicated with perivalvular abscess  with extensive debridement of the infected and necrotic tissues surrounding the aortic annulus  at night that persisted for 3 days. He had previously undergone 25-mm mechanical aortic valve implantation  12 years prior because of severe aortic regurgitation. Upon examination, no focus of infection could be identified, and he was admitted to our heart centre to exclude the possibility of PVE. After three sets of blood culture were obtained, intravenous ceftriaxone (2 g/day) was empirically initiated. Initial transthoracic echocardiography (TTE) revealed no dysfunction of the mechanical aortic prosthesis, and whole-body CT revealed no embolic events. The patient recovered to a low-grade fever (37–38°C) 48 h after the initial antibiotics therapy and showed improved C-reactive protein level (from 13 to 6.3 mg/dL) in the first 5 days. Subsequently, transoesophageal echocardiography (TOE) performed on the 5th day of hospitalization revealed a 6–11 mm mobile echogenic mass attached to the prosthetic valve and a thickened area within the posterior annular region with a low echogenic appearance at the level of the non-coronary sinus of Valsalva , Enterococcus spp. and Streptococcus bovis were reported to be the most common pathogens in patients with PVE. We carefully selected a combination of ampicillin and gentamicin to mainly treat staphylococci, streptococci, and enterococci. We assumed that the patient’s clinical course was relatively subacute and less destructive with no major complications such as heart failure and systematic embolism and ceftriaxone seemed to be clinically effective. Considering this, PVE triggered by Staphylococcus aureus or CNS was slightly less likely to occur in the patient. Blood culture-negative infective endocarditis (BCNIE) can be caused by fungi or fastidious slow-growing bacteria, notably obligatory intracellular bacteria.Streptococcus spp. and HACEK  could be the potential causative microbes in the present case; therefore, we selected a combination of ampicillin and gentamicin for treatment based on the local epidemiology. Indeed, we were ready to perform systemic serological testing for less common microorganisms in BCNIE patients, such as Coxiella burnetii, Bartonella spp., Aspergillus spp., Mycoplasma pneumonia, Brucella spp., and Legionella pneumophila, followed by specific PCR assays. However, we kept the samples and did not perform these tests because the patient’s condition had gradually recovering after the initiation of intravenous ampicillin and gentamicin during the whole hospital stay, including the perioperative period. Additionally, both the blood and tissue PCR tests, which are not covered by Japan’s national health insurance, generally take a considerable amount of time and cost. A 6-month outpatient oral antibiotic therapy was completed because we emphasized on the remarkable findings: PVE was complicated with perivalvular abscess formation and unidentified microorganisms.Another important case discussion was on how to manage the blood culture-negative patient with PVE with appropriate antibiotic therapy and/or surgical intervention if needed. The patient was initially treated with ceftriaxone in empirical therapy for unexplained fever following three sets of blood culture tests. Although the patient’s condition stabilised in the first week, with improvement in fever and general appearance, we switched ceftriaxone to intravenous ampicillin and gentamicin because the findings of 18F-FDG PET/CT successfully provided a definitive diagnosis of perivalvular abscess and infective endocarditis, leading to subsequent surgical intervention within an appropriate timeframe.In summary, we report a case regarding a patient with blood culture-negative PVE complicated with perivalvular abscess, with a low-to-intermediate likelihood of PVE based on the modified Duke criteria, in whom Dr Shiro Miura is working as a chief interventional cardiologist at one of the most outstanding cardiovascular centers in Hokkaido in Japan named Hokkaido Ohno Memorial Hospital with remarkable achievements specializing in interventions on ischemic heart diseases and structural heart disease in the management of inpatients and outpatients with heart failure following the postgraduate course named ‘MSc Statistics with Applications in Medicine’ in University of Southampton in the UK where I was rewarded the Dean’s List Awards for exceptional performance for both the regular term exams and final dissertation.European Heart Journal - Case Reports online.Slide sets: A fully edited slide set detailing this case and suitable for local presentation is available online as Consent: The author/s confirm that written consent for submission and publication of this case report including image(s) and associated text has been obtained from the patient in line with COPE guidance. Conflict of interest: none declared.ytz171_Supplementary_Slide_SetClick here for additional data file.


LINE-1) and DNA methylation status of the PlGF gene.The pathophysiology of preeclampsia is largely unknown. Serum placental induced growth factor (PlGF) levels are decreased during second trimester pregnancy. Aberrant DNA methylation is suggested to be involved in the etiology of preeclampsia (PE). We hypothesize that DNA methylation is altered in PE placentas determined the methylation index by measuring placental S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels. In addition, we assessed global DNA methylation status by long-interspersed nuclear element-1  corrected for gestational age and birth weight.Placental tissue of 11 early onset PE (EOPE), 11 late onset PE (LOPE) and 60 controls consisting of 25 uncomplicated controls 20 fetal growth restriction (FGR) and 15 preterm births (PTB) controls was collected from a nested case-control study of The Rotterdam Periconceptional Cohort. RNA and DNA was isolated from placental tissue and DNA was treated with sodium bisulfite. SAM and SAH levels were measured by LC-ESI-MS/MS. Methylation of PlGF DNA methylation was decreased in placental tissue of EOPE cases versus LOPE , uncomplicated controls , FGR controls  and PTB controls (mean difference -11.3 ± 3.8% P = 0.04). No significant differences were observed in SAH, SAM:SAH ratio, LINE-1 DNA methylation and PlGF mRNA expression between groups.Placental SAM levels were significantly lower in placental tissue of EOPE pregnancies compared to PTB controls . PlGF DNA hypomethylation underlines the possible role of placental DNA hypomethylation in the pathophysiology of EOPE, which needs further investigation.The hypomethylation state of the placenta in EOPE, which is reflected by lower SAM and  Preeclampsia (PE) is one of the most severe maternal pregnancy complications worldwide and affects 2–8% of all pregnancies . The patLINE-1 repetitive elements) and decreased methylation of imprinted genes . W. W23]. W studies –29. HowePlGF DNA methylation, which suggests that less methyl donor availability from SAM leads to decreased PlGF DNA methylation. More studies with larger sample size are necessary to evaluate the potential role of SAM in PE.Based on the previous described role of altered one-carbon metabolism in relation to PE we hypothesized that changes in SAM, SAH and SAM:SAH ratio are present in PE, which lead to decreased methylation. We showed significantly lower SAM levels in placental tissue of EOPE pregnancies compared to PTB controls, which result in a lower SAM:SAH ratio, although this ratio was not significant (P = 0.13). These lack of significant lower SAM:SAH ratio might be caused by the relative small sample size of our study. SAM:SAH ratio has been described as the methylation potential of the cell and lower levels of SAM or are expected to be associated with less methylation. In addition, we did find a modest positive correlation of placental SAM levels with LINE-1 methylation between PE pregnancies and controls. Not many studies have investigated the role of global methylation measured by LINE-1 repeats in preeclampsia. Placental LINE-1 DNA hypermethylation was found in two studies [LINE-1 methylation was not significantly changed in PE compared to controls [LINE-1 methylation in placental tissue of PE complicated pregnancies is not different from control pregnancies. However, more studies are necessary to draw a firm conclusion.We were not able to demonstrate differences in placental  studies , 31 wher studies , 32. In controls –35, whicPlGF in PE complicated pregnancies and compared this to uncomplicated pregnancies and pregnancies complicated by FGR and PTB. In most studies, DNA methylation of PE complicated pregnancies was compared to uncomplicated controls. However, uncomplicated controls differ significantly from PE complicated pregnancies in gestational age and birth weight. To circumvent this we included FGR-, and PTB complicated controls, which are more comparable in birth weight and gestational age to PE complicated pregnancies. However, despite including these control groups we still observed significant differences in birth weight and gestational age between PE complicated pregnancies and PTB- and FGR complicated controls for which we corrected. In addition, we found a modest negative correlation between PlGF DNA methylation and SAM levels with gestational age. In addition, SAM was also correlated to birth weight. These findings demonstrate that correction for birth weight and gestational age is needed to assess independency and also underlines that these methylation levels change during pregnancy, which warrant further studies during second trimester pregnancy.In our study we assessed placental methylation status of PlGF DNA hypomethylation in placental tissue from EOPEpregnancies compared to each of the control groups, which was not present in LOPE. The underlying mechanism in EOPE and LOPE differs. It has been suggested that in EOPE poor placentation plays a dominant role, whereas in LOPE inflammatory processes are involved [PlGF DNA hypomethylation in EOPE but not in LOPE [We demonstrated involved . Studies in LOPE , 37.PlGF DNA hypomethylation in EOPE is associated with lower PlGF mRNA expression we quantified mRNA expression in placental tissue but did not find evidence for changes in PlGF mRNA levels between EOPE, LOPE, and each of the control groups, which is in line with a previous study which showed that PlGF mRNA expression was not altered in EOPE placentas [To assess whether the observed lower lacentas .PlGF DNA methylation was negatively correlated to PlGF mRNA expression in uncomplicated controls, whereas in pregnancies complicated by EOPE, LOPE, FGR or PTB no significant correlation was observed. This suggests that under physiological circumstances PlGF mRNA expression might be regulated by PlGF methylation. However, under pathophysiological circumstances, such as EOPE, different regulation of PlGF methylation and mRNA expression occurs, which need to be further examined in future studies.Interestingly, we did find that PlGF DNA methylation was not associated with PlGF mRNA expression, which could have the following explanations; 1) PlGF promoter DNA methylation does lead to changes in PlGF mRNA expression, 2) due to large variation in PlGF mRNA expression we were not able to detect subtle differences in mRNA expression, 3) the physiological mechanism of PlGF downregulation at birth might differ from second trimester pregnancy. Future studies investigating PlGF DNA methylation in maternal plasma or chorionic villi obtained during second trimester pregnancy will be needed to assess whether the lower PlGF DNA methylation levels can be used to predict EOPE in the near future.During second trimester pregnancies lower PlGF plasma levels have been observed in EOPE pregnancies. In our study PlGF DNA methyalation in preeclampsia. We showed lower SAM and PlGF DNA hypomethylation in placental tissue of EOPE complicated pregnancies compared to PTB controls. However as changes in methylation markers were modest more studies are needed to validate our findings and to assess the clinical value in relation to prediction of EOPE during first-trimester pregnancy.This is the first study that assessed placental DNA methylation of global markers of methylation and