Mechanical ventilation is a resource intensive organ support treatment, and historical studies from low-resource settings had reported a high mortality. We aimed to study the outcomes in patients receiving mechanical ventilation in a contemporary low-resource setting.We prospectively studied the characteristics and outcomes  in 237 adults mechanically ventilated for a medical illness at a teaching hospital in southern India during February 2011 to August 2012. Vital status of patients discharged from hospital was ascertained on Day 90 or later.P = 0.001) and ventilator-associated pneumonia . But, their mortality was significantly lower compared to the rest .Mean age of the patients was 40 ± 17 years; 140 (51%) were men. Poisoning and envenomation accounted for 98 (41%) of 237 admissions. In total, 87 (37%) patients died in-hospital; 16 (7%) died after discharge; 115 (49%) were alive at 90-day assessment; and 19 (8%) were lost to follow-up. Weaning was attempted in 171 (72%) patients; most patients (78 of 99 [79%]) failing the first attempt could be weaned off. Prolonged mechanical ventilation was required in 20 (8%) patients. Adherence to head-end elevation and deep vein thrombosis prophylaxis were 164 (69%) and 147 (62%) respectively. Risk of nosocomial infections particularly ventilator-associated pneumonia was high . Higher APACHE II score quartiles , and new-onset organ failure (2.98 [1.94–4.56]) were independently associated with the risk of death. Patients with poisoning had higher risk of reintubation (43% vs. 20%; The case-mix considerably differs from other settings. Mortality in this low-resource setting is similar to high-resource settings. But, further improvements in care processes and prevention of nosocomial infections are required. Mechanical ventilation is an important organ support treatment given to patients admitted in intensive care units (ICUs). Apart from requiring specialised equipment and logistics, trained healthcare personnel are also needed to provide care to mechanically ventilated patients. Availability of all these elements is essential for effective care delivery. But, there is a marked global disparity in healthcare resources  available to deliver ICU care including mechanical ventilation ,2,3. HenApart from disparities in resources, the pattern of critical illnesses encountered in such settings is likely to substantially differ from the developed world. Therefore, it is essential to know whether the use of mechanical ventilation in low-resource settings is clinically effective. The outcomes of mechanical ventilation have been characterised in large unselected multinational patient populations from the developed world . These sThis is a prospective hospital-based case series study of eligible patients receiving mechanical ventilation. The study protocol was reviewed and approved by the Institute Ethics Committee (Human studies) at Jawaharlal Institute of Postgraduate Medical Education and Research (JIPMER), Puducherry . The study was conducted at the Medical ICU of JIPMER Hospital during the period February 2011 to August 2012. This is a government-run teaching hospital located in the southern Indian town of Puducherry providing free-of-charge primary as well as referral care services to patients from the union territory of Puducherry and surrounding regions of the state of Tamil Nadu. In the Medical ICU, we take care of critically-ill non-surgical patients aged more than 12 years who are transferred from the emergency department and hospital floors. This is an eight-bedded ICU manned exclusively by the Department of Medicine. During the study, this ICU switched from an ‘open’ to ‘closed’ pattern of care led by a full-time internal medicine consultant during the daytime since July 2011. Postgraduate residents in their second and third years of residency program are posted in the ICU on 12-hour work shifts for a total duration of 2–3 months. The nurse-to-patient ratio of this ICU is 1:4.Patients were included in the study after obtaining informed written consent from the next of kin. Patients were eligible for inclusion if they were aged more than 12 years, had a medical-illness as the cause of hospitalization, and were receiving mechanical ventilation. Patients were excluded if they had any one of the following—patients transferred intubated from another hospital; patients receiving mechanical ventilation for < 24 hours; baseline evaluation could not be performed in the first 24 hours following intubation; or patients on non-invasive ventilation.2/FiO2 was ≤ 300; hematologic failure if platelet count was ≤ 100,000/μL (≤ 100×109/L); hepatic failure if bilirubin was ≥ 2 mg/dL (≥ 34.2 μmol/L); circulatory failure if they required any dose of vasopressor/inotrope; neurological failure if Glasgow coma score was < 13; and renal failure if the serum creatinine was ≥ 2 mg/dL (≥ 176.8 μmol/L).The following parameters were recorded at the baseline evaluation—age, gender, smoking status, alcohol abuse, primary diagnosis, severity of illness scores , and presence of co-morbidities such as diabetes, hypertension, and asthma. Laboratory parameters including blood counts, serum creatinine, electrolytes, liver function tests, and arterial blood gas analysis were also noted at this evaluation. Presence of organ failure at baseline was defined using the sequential organ failure assessment (SOFA) definitions —patientsPatients were followed-up during hospital stay and the outcomes were assessed in three domains—disease-related, mechanical ventilation-related, and process of care-related outcomes. Disease-related outcomes assessed were in-hospital mortality, 90-day mortality, and new-onset organ failure. The same cut-offs as above were used to define new-onset organ failure. The 90-day survival was assessed through telephonic inquiry. Following hospital discharge, patients or their family members were contacted over the telephone on Day 90 (from the day of intubation) or later to assess the vital status of the patient. If the patient could not be contacted even after multiple attempts, then the patient was adjudicated as lost to follow-up.Mechanical ventilation-related outcomes studied were duration of ventilation, timing of tracheostomy, barotrauma, endotracheal tube block, unplanned extubation, weaning failure, and extubation failure. Prolonged mechanical ventilation was defined as ventilation for > 21 days . WeaningProcess of care-related outcomes studied were deep vein thrombosis (DVT), pressure sores, nosocomial infections, and adherence to standard of care practices . In addition to DVT confirmed by compression ultrasound, clinically suspected but unproven episodes were also considered as incident DVT. Ventilator-associated pneumonia (VAP) was defined as both confirmed as well as suspected episodes of VAP as diagnosed by the treating physician that necessitated treatment with antibiotics.Data on in-hospital mortality and new-onset organ failure were prospectively collected from case records. We anticipated that data for some outcomes such as endotracheal tube block and unplanned extubation might not be reliably captured in case records. Hence, in addition to perusing case records, the care providers were also interviewed to obtain this information. Documentation of nosocomial infections in case records could suffer from underreporting bias for various reasons. To avoid this, we used much broader criteria to adjudicate on VAP—while this is expected to be very sensitive, its specificity for VAP diagnosis is likely to be modest. Adherence to head-end elevation and presence of pressure sores were assessed by the investigator during data collection visits. All data were collected by the same investigator (BK) ensuring uniformity.Since published estimates of 90-day mortality from a similar setting were unavailable to inform the sample size calculation, we assumed the 90-day mortality to be 50%. To estimate this within 7 percentage points of the true value (absolute precision) with 95% confidence, we needed to study 196 patients . AllowinContinuous variables with an approximate normal distribution are summarized as mean ± SD or as median  when the distribution is skewed. Categorical variables are presented as frequency with proportions, n (%). We present 95% confidence intervals for point estimates of mortality, and describe the risk of nosocomial infections as incidence-density. We used the Kaplan-Meier survival plot to depict the survival distribution of patients, and compared the survival curves between groups by log-rank test. While analysing risk factors for mortality, we excluded patients that were lost to follow-up and that left care against medical advice. We compared the continuous variables between survivors vs. non-survivors by independent sample t-test or Wilcoxon rank-sum test as appropriate, and by chi-square test for categorical variables. We assessed the impact of case-mix by comparing the subgroup of acute poisoning patients with the rest.P < 0.1 threshold on univariate analysis were considered for entry into the model. To avoid overfitting, we built a parsimonious model with the least number of explanatory variables ) could be weaned off and discharged from hospital; 10 of them needed prolonged ventilation beyond Day 21. The remaining 21 patients failing the first weaning attempt died in the hospital (20 patients) or left against medical advice (1 patient); 8 of these 21 patients required prolonged mechanical ventilation . Fifty (One hundred and forty seven (62%) patients received DVT prophylaxis—enoxaparin 135 (57%); unfractionated heparin 11 (5%); and elastic compression stockings 1 patient. Forty-nine (21%) patients had contraindications for pharmacological prophylaxis. In total, four (2%) patients developed lower limb DVT —two had In total, 147 (62%) patients developed at least one nosocomial infection. The details of nosocomial infections are presented in Of the 237 patients, 87 (37% [95% CI 31%- 43%]) died in the hospital; 147 (62% [56%- 68%]) survived to hospital discharge. The median duration of follow-up for the survivors was 150 (101–203) days. Sixteen patients (7% [4%-11%]) died at home during the follow-up period; and 115 (49% [42%- 55%]) patients were alive on Day 90 or later . DetailsP < 0.001). There was a progressive increase in the risk of mortality in the upper APACHE II quartiles compared to the lowest quartile (P < 0.001). Likewise, patients with new-onset organ failure had about 3.5-fold risk of mortality (P < 0.001). When cross-classified, patients with new-onset organ failure in addition to other organ failure at baseline had the least chances of survival; whereas, patients without any organ failure throughout had the best chances of survival , longer duration of mechanical ventilation , higher PEEP  and FiO2 requirements , higher plateau pressure , lower platelet count , and lower hematocrit .Apart from these factors, age, serum albumin, diabetes, and hypertension were also associated with mortality on univariate analysis . After aThe subgroup of patients with acute poisoning was significantly younger and more likely to be male . Co-morbWe found that the case-mix of patients receiving mechanical ventilation for medical-illness in our setting was dominated by acute poisoning, and this had a bearing on the clinical outcome in these patients. We also found that, notwithstanding the limited resources, the mortality was similar to that documented elsewhere. But, the care practices and outcomes miss the benchmark in some key areas such as adherence to ventilator bundle and risk of nosocomial infections. Further, this study also uncovered that a considerable proportion of patients die after hospital discharge.The current study has several strengths—i) the sample size was large enough to obtain precise estimates of mortality and to examine the risk factors for 90-day mortality; ii) data collection was prospective; iii) the study sample was not restricted to any particular diagnostic group; iv) a holistic assessment of outcomes was done in three domains; and v) the loss to follow-up was minimal. On the other hand, the present study has some limitations. First, we did not attempt to verify the accuracy of a diagnosis of VAP. Notwithstanding, this metric reflects the real life situation, at least in our setting. Second, the follow-up period was comparatively short to assess post-discharge mortality. Nonetheless, to our knowledge, this is the first study from India to provide an empirical estimate of the post-discharge mortality. Third, we did not study the use of sedation, neuromuscular blockers, and nutritional support.In the present study, poisoning and envenomation accounted for nearly 40% of ICU admissions requiring mechanical ventilation. This is strikingly different from developed countries where most of the admissions were due to pneumonia, chronic obstructive pulmonary disease, sepsis, heart failure, and neurological diseases ,12. ThisIn the present study, the in-hospital mortality was 37%. Esteban et al found that the in-hospital mortality of mechanically ventilated patients had improved from 40% to 35% over the period of 1998 to 2010 in three multinational cohorts . Thus, tThe mean duration of mechanical ventilation in the present study was longer compared to the findings of the MV-ISG (10.6 vs. 5.9 days) ,21. InfrWe found that most patients received DVT prophylaxis, and 17% patients did not receive DVT prophylaxis despite having no contraindication. In the multinational ENDORSE study, only 40% of at-risk medical patients received DVT prophylaxis . The adhRisk of nosocomial sepsis, especially VAP, was high in our ICU. The incidence was very high compared to developed nations such as the United States and Scotland where it is 1.0 and 6.5 per 1,000 ventilator-days respectively ,26. HoweThe present study indicates that mechanical ventilation could be clinically effective in low-resource settings. But, further improvements in care processes and prevention of nosocomial infections are necessary, and this calls for commitment of more resources to ICU services. The findings of the present study would be applicable to settings where a similar case-mix is encountered. Even when the case-mix differs, our findings might be applicable to low-resource settings with material resources for mechanical ventilation, but not specialist doctors, nurses, and other paramedical personnel. But, it is unclear whether the present findings could be generalised to non-teaching hospitals in low-resource settings.Acute poisoning dominates the case-mix of patients receiving mechanical ventilation in our setting. Characteristics of this subgroup of patients distinctly differ from the rest, and their prognosis is better. Moreover, the difference in case-mix explains at least partly the observed differences in outcomes such as reintubation and VAP. Finally, despite the resource limitations, the overall survival is similar to other settings. However, process of care measures such as adherence to ventilator bundle and prevention of nosocomial infections need concerted efforts for improvement.S1 FigAMA = Left against medical advice; PMV = prolonged mechanical ventilation; SBT = Spontaneous breathing trial.(TIF)Click here for additional data file.S2 FigHash marks on the plot indicate censoring. In two patients follow-up was censored at 70 days and 83 days.(TIF)Click here for additional data file.S3 FigHash marks on the plot indicate censoring. In two patients follow-up was censored at 70 days and 83 days.(TIF)Click here for additional data file.S1 FileDe-identified minimal dataset.(XLS)Click here for additional data file.S1 TableAPACHE II = Acute physiology and chronic health evaluation II.(DOCX)Click here for additional data file.


Alzheimer disease (AD) is the most common form of dementia but the identification of reliable, early and non-invasive biomarkers remains a major challenge. We present a novel miRNA-based signature for detecting AD from blood samples.We apply next-generation sequencing to miRNAs from blood samples of 48 AD patients and 22 unaffected controls, yielding a total of 140 unique mature miRNAs with significantly changed expression levels. Of these, 82 have higher and 58 have lower abundance in AD patient samples. We selected a panel of 12 miRNAs for an RT-qPCR analysis on a larger cohort of 202 samples, comprising not only AD patients and healthy controls but also patients with other CNS illnesses. These included mild cognitive impairment, which is assumed to represent a transitional period before the development of AD, as well as multiple sclerosis, Parkinson disease, major depression, bipolar disorder and schizophrenia. miRNA target enrichment analysis of the selected 12 miRNAs indicates an involvement of miRNAs in nervous system development, neuron projection, neuron projection development and neuron projection morphogenesis. Using this 12-miRNA signature, we differentiate between AD and controls with an accuracy of 93%, a specificity of 95% and a sensitivity of 92%. The differentiation of AD from other neurological diseases is possible with accuracies between 74% and 78%. The differentiation of the other CNS disorders from controls yields even higher accuracies.The data indicate that deregulated miRNAs in blood might be used as biomarkers in the diagnosis of AD or other neurological diseases. Alzheimer disease(AD) is the most common form of neurodegenerative illness leading to dementia which is predicted to affect as much as 1 in 85 people globally by 2050 .A prominent example of recently developed AD biomarker assays is the combinatorial analysis of the concentration of peptides and proteins: beta-amyloid-1-42 (Aß 42), tau, and/or p-tau in the cerebrospinal fluid (CSF). According to the S3 guidelines, an increased level of tau protein together with a decreased level of beta-amyloid-1-42 provides strong evidence for the presence of AD . FurtherThe use of peripheral markers, like Aß and tau in easily accessible peripheral cells (in particular platelets and skin fibroblasts), as a diagnostic tool has been under investigation for more than 10 years ,8. MolecThere is increasing effort to develop molecular diagnostic markers that meet requirements like easy accessibility, for example, from blood, sufficiently high specificity and sensitivity, low costs and applicability by laboratories with standard equipment. Several blood, plasma, or serum born AD biomarkers have been proposed to meet these criteria. Doecke et al. recently presented a panel of protein biomarkers to reliably detect AD with an accuracy of 85% . Moreovein vitro diagnostic testing by molecular diagnostics and CLIA  laboratories.Besides proteins microRNAs (miRNAs) have also demonstrated their potential as non-invasive biomarkers from blood and serum for a wide variety of human pathologies . A deregWhile altered miRNA patterns have been exhaustively investigated in AD patients' tissue samples or cell cultures -18, lessWe describe our results obtained by applying the next-generation sequencing (NGS) approach to screen the expression of all human miRNAs in blood from extensively characterized AD patients and healthy controls. Patient blood was obtained from the SAMPLE  Registry of PrecisionMed  and blood from age-matched healthy donors from the ACE  Registry, a PrecisionMed- UBC (The University of British Columbia) collaboration. We identified 140 unique differentially expressed miRNAs between AD patients and controls. Validation of a 12-miRNA signature was carried out by RT-qPCR in a cohort of 202 samples encompassing patients suffering from other neurological disorders including mild cognitive impairment as a potential preliminary stage of AD, and other neurodegenerative diseases like Parkinson disease and multiple sclerosis as well as mental diseases like schizophrenia (SCHIZ), major depression (DEP), and bipolar disorder (BD).A combination of AD-specific miRNA expression signatures with the rapidly developing and expanding amyloid load imaging techniques may be useful as non-invasive diagnostic tools in AD diagnosis in the future .To detect potential AD biomarkers we examined blood from well-characterized patients and controls. We obtained blood from the SAMPLE  Registry of PrecisionMed . SAMPLE is a sample depository resulting from a longitudinal study that evaluates cognition in women and men, who are recruited, evaluated, cognitively studied, and sampled from 12 to 15 experienced investigative sites in USA. All participants underwent several tests , Clinical Dementia Rating (CDR), Wechsler Memory Scale, and Mini-Mental State Exam (MMSE)) to evaluate cognition. Blood from age-matched healthy donors was obtained from the Ace Registry, which is a biological sample bank of serial patient samples with linked serial cognition data, based on a cognition battery selected from UBC's proprietary computerized testing platform.We carried out high-throughput NGS of 22 healthy control samples (C) and 48 AD patient samples using IlluminaHiSeq 2000 sequencing with eight multiplexed samples on each sequencing lane. We detected not only known human miRNAs, but also novel miRNA candidates that have previously not been included in the miRBase v18 ,22. ThesPvalue of 8.35*10-6and the most downregulated miRNA was hsa-miR-144-5p (AUC of 0.9138) with Pvalue of 8.35*10-6. While the high AUC value indicates that each of these miRNAs has sufficient power to differentiate between AD and healthy controls, they are not specific for AD since both miRNAs have already been described for many other human pathologies, including different neoplasms [Pvalue <0.05 after adjustment for multiple testing), with the interesting categories miR-30 family with five miRNAs being upregulated , the let-7 family with nine downregulated miRNAs , and the disease category Alzheimer disease for which six dys-regulated miRNAs were relevant, including hsa-miR-21, hsa-miR-17, hsa-miR-29a, hsa-miR-29b, hsa-miR-106b, and hsa-miR-107 .To compare the NGS results of the AD patient samples with the samples from healthy donors we first computed Wilcoxon-Mann-Whitney (WMW) test and adjusted the significance values for multiple testing using Benjamini-Hochberg adjustment. All miRNAs with adjusted significance values <0.05 were considered statistically significant. We also computed the area under the receiver operator characteristics curve (AUC). In total, we detected 180 significantly dys-regulated miRNAs (140 unique mature miRNAs) including 90 miRNAs (58 unique mature miRNAs) that were downregulated and 90 miRNAs (82 unique mature miRNAs) that were upregulated in AD samples compared to healthy control samples , we extracted potential TF binding sites from the UCSC genome browser but found no evidence for a significant enrichment for specific TF binding sites.In the next step, we performed classification of AD and control samples using a standard machine learning approach. In a cross-validation loop, we stepwise added the miRNAs with lowest significance values and repeatedly carried out radial basis function support vector machines (SVM). As shown in Figure To validate the 12-miRNA signature we employed RT-qPCR and included not only additional patients with AD, but also patients with other diseases including neurological disorders. In total, we analyzed 12 miRNAs in 202 samples as detailed in Table We first considered the miRNA fold quotients that were obtained for AD samples and controls. We compared the fold quotients of each of the 12 miRNAs between initial NGS screening cohort and the RT-qPCR validation cohort. All but two of the 12 miRNAs, namely hsa-miR-1285-5p and hsa-miR-26a-5p, have been dys-regulated in the same direction in both approaches, indicating a high degree of concordance between screening and validation study. Both hsa-miR-1285-5p and hsa-miR-26a-5p have been significantly upregulated in AD in the NGS screening experiment while downregulated in the RT-qPCR validation  and moderate AD . The MMSE is a short test of 30 questions used to screen for cognitive impairment. Each question to be answered is scored with points, with a maximum possible score of 30 points. This questionnaire can be used to estimate the severity of cognitive impairment and to follow the course of cognitive changes in an individual over time. Normally, patients reaching 27 to 30 points do not suffer from dementia, 10 to 26 points are indicative for mild-to-moderate dementia, and less than 9 points indicates severe dementia. We found no significant expression differences of the 12-miRNA signature between the mild AD group and the moderate AD group.To evaluate whether the selected miRNAs are stage-dependent we further grouped the AD patients according to their MMSE score into mild AD , multiple sclerosis ) and patients with psychiatric disorders  for the signature of 12 miRNAs. The pattern, which was closest to AD was SCHIZ, where we found six up- and six downregulated miRNAs. We found a strong overall downregulation for most of the selected 12 miRNAs for patients with DEP and PD and a strong overall upregulation for patients with MCI, CIS, and BD Figure .versus control and neurodegenerative and psychological disorders versus control) reached values of 84.2% and 86.1%, respectively. Furthermore, we tested how well the 12-miRNA signature separates AD from MCI, AD from psychological disorders, and AD from other neurodegenerative diseases, respectively. The average accuracy for these comparisons was between 73.8% and 77.8%. Since the 12-miRNA signature has been tailored to differentiate between AD and controls, other miRNAs may likely contribute to a signature that permits also a better differentiation between the other tested diseases and AD.In addition, we also applied machine learning procedures using SVM to estimate the accuracy, sensitivity, and specificity of the 12-miRNA signature regarding the other neurological diseases in comparison to the control group and to AD. The results of these classifications are also listed in Table Pvalues <0.05 and FDR adjustment. Interestingly, we found a significant enrichment of miRNA targets in the GO categories nervous system development, neuron projection, neuron projection development, and neuron projection morphogenesis. These GO categories are listed in Table Target gene prediction of the 10 known miRNAs from the 12-miRNA signature revealed 2,354 genes that may be regulated by those miRNAs. These target genes were used to perform an over-representation analysis and identified 73 computed Gene Ontology (GO) categories with Pvalue <0.05 for brain-miR-112, with significant enrichment of miRNA targets in GO categories associated with nervous system and neuron function  yields too low diagnostic accuracy and is therefore not recommended as diagnostic test for AD. Furthermore, Ray et al. yielded promising results by the identification of 18 proteins in blood plasma that could differentiate AD patients from controls with 90% accuracy .Here, we investigate whether blood-borne miRNA expression signatures might contribute to AD diagnosis. Until now, many efforts have been made to understand the role of miRNAs in neurodegenerative disorders, as summarized by Eacker et al. . HoweverUsing high throughput sequencing, we identified 140 unique miRNAs from 180 precursors that were differentially expressed between whole blood obtained from AD patients and healthy controls. It is incumbent upon the investigator, who proposes a set of miRNAs as done here to examine whether there is any known connection of these miRNAs and their target genes to neurodegeneration. Below we discuss this aspect in respect to our findings of dys-regulated miRNAs in blood of AD patients compared to healthy controls.in vitro. Furthermore, they found a statistically significant decrease in hsa-miR-106b expression in sporadic AD patients, but the correlation between miR-106b and APP expression in AD brain was not significant [in vitro and under physiological conditions in cells [According to our TAM analysis out of the downregulated miRNAs, six were associated with the disease category Alzheimer disease including hsa-miR-21, hsa-miR-17, hsa-miR-29a, hsa-miR-29b, hsa-miR-106b, and hsa-miR-107. In a mouse model, Wang et al. investigated the involvement of hsa-miR-106b in the TGF-β signaling pathway that plays a key role in the pathogenesis of AD and found an inverse correlation between the expression of hsa-miR-106b and TGF-β type II receptor (TβR II) protein level . In addinificant . The samnificant . Shioya nificant . They alnificant . Hsa-miRin cells ,33. MiR-in cells .We further performed over-representation analysis with the 2,354 predicted targets of the 10 known miRNAs of our 12-miRNA signature. Here, several GO categories, with significant enrichment of miRNA targets in the GO categories linked to the nervous system, were found. Most interestingly, some of these target genes have already been related to AD or other of the investigated neurological diseases. One of the most prominent examples is DRD1 that encodes the Dopamine receptor D1, which is the most abundant dopamine receptor in the central nervous system. DRD1 is associated with AD, BD, and SCHIZ. Another example, DISC1 (disrupted in SCHIZ), associated with BD and SCHIZ, encodes a protein involved in neurite outgrowth and cortical development. BDNF (brain-derived neurotrophic factor) important for survival of striatal neurons in the brain is known to be downregulated in AD patients and also associated with BD, DEP, PD, and SCHIZ. IGF1R is the only target gene that was exclusively found to be associated with AD. The protein encoded by this gene is increased in temporal cortex surrounding and within Aß-containing plaques, but a significantly lower number of neurons of AD patients express IGF1R -38. AnotHowever, we have to point out that our analysis is based on whole blood. Previous findings on cancer suggest that the miRNA expression pattern between blood cells and cancer tissue do not necessarily show the same expression pattern but some overlaps can be found -42. UnfoAPP and the formation of the amyloid plaques. Database analyses prompted them to further investigate two miRNAs that were also included in our 12-miRNA signature, namely hsa-miR-107 and hsa-miR-103. They found that predicted target genes of hsa-miR-107 and hsa-miR-103 showed significant overlap with the AlzGene database. In a reporter assay ADAM10 expression was reduced by both miRNAs. These two miRNAs were also investigated in relation to the expression of cofilin protein in a transgenic mouse model [Since a large set of miRNAs often contains a significant amount of redundant miRNAs with largely identical information content the differentiation between AD samples and healthy controls using a reduced set of miRNAs may likely yield comparably accurate results. Therefore, a panel of 12 miRNAs with limited cross-correlation, including most strongly dys-regulated miRNAs that show a potential to separate AD from controls, was selected. Some of these 12 miRNAs have already been related to AD. For example, Wang et al. showed in a computational analysis that the 3'-untranslated region (UTR) of beta-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA is targeted by hsa-miR-107 and that BACE1 mRNA levels tended to increase as miR-107 levels decreased in the progression for AD. An increased BACE1 expression is an important risk factor for sporadic AD . Nelson se model . Cofilinversus controls yielded an even better accuracy than for AD versus controls. These findings suggest a relevance of the considered 12 miRNAs also for psychological disorders. The association of the 12-miRNA signature with neurological diseases in general is further underlined by the results of our over-representation analysis using GeneTrail. Here, we found four significant GO categories related to nervous system and neurons with an over-representation of target genes of the 10 known miRNAs from our 12-miRNA signature. In addition, out of the 10 known miRNAs nine miRNAs are already included in the HMDD and five of those miRNAs that were previously associated with neurological diseases including AD, PD, and SCHIZ. As mentioned above, Yao et al. [While we showed the 12-miRNA signature's potential to separate AD patients from controls with an accuracy of 93.3%, we also tested its applicability as differential diagnostic biomarker to separate AD from other neurological diseases. As we expected, the accuracy decreased when trying to use this signature for separating other neurodegenerative diseases from controls or separating AD from other neurological disorders. Remarkably, the classification of psychiatric disorders o et al.  showed to et al.  showed to et al. . Target o et al. , NLGN1 . The age distribution of AD patients, MCI patients, PD patients, and controls is quite similar. Patients suffering from multiple sclerosis, DEP, BD, or SCHIZ are about 20 to 30 years younger. The differences in the age distribution are due to the differences between the onsets of the diseases. In previous studies [Pvalue was hsa-miR-423 . To test for the influence of age we compared the profiles obtained from old versus young patients by splitting the total group in half based on the age. Here, the miRNA with the lowest Pvalue was hsa-miR-890 . Again, we did not find any deregulated miRNAs. In summary, we found no evidence that age and gender have a substantial influence on the miRNA profiles. Both miRNAs mentioned above were not significant in the present study on AD.Most importantly one needs to point out that as the patients included in our study are not treatment-naïve, we cannot exclude the influence of administered drugs on the miRNA signature. As an example, Bocchio-Chiavetto et al. showed that chronic anti-depressant treatment has effects on the blood miRNA profile . Further studies  we alreaHere we identified 140 unique differentially expressed miRNAs between AD patients and healthy controls. Using a signature of 12 miRNAs differentially expressed between AD patients and healthy controls we were not only able to distinguish with high diagnostic accuracies between AD patients and healthy controls, but also between AD patients and patients suffering from other neurological disorders including mild cognitive impairment as a potential preliminary stage of AD, and other neurodegenerative diseases like PD and multiple sclerosis as well as mental diseases like SCHIZ, DEP, and BD. However, additional work will be needed to elucidate the applicability of this 12-miRNA signature as a potential diagnostic test for AD and the above-mentioned effects of the drug treatments commonly used in the treatment of the disease. Hopefully, tests of this non-invasive and relatively cheap kind will be applicable to prodromal AD cases and to MCI patients with the aim to recognize early AD to initiate treatment.n = 106), patients with mild cognitive impairment (MCI) (n = 18), patients with multiple sclerosis  (n = 16), patients with PD (n = 9), patients with DEP (n = 15), patients with BD (n = 15), patients with SCHIZ (n = 14), and from healthy controls (C) (n = 22). Samples from patients with AD stem from the Biorepository and Tissue Bank PrecisionMed  (n = 97) and the University Clinic of Erlangen (Germany) (n = 9), samples from healthy controls and from patients with MCI, PD, DEP, BD, and SCHIZ stem from PrecisionMed  and samples from patients with CIS stem from Charité Berlin (Germany). Detailed patient characteristics are listed in Additional file We analyzed the expression of miRNAs in peripheral blood of a total of 215 patients and healthy controls, either by NGS or by RT-qPCR or by both methods , abnormal memory function documented by scoring below the education adjusted cutoff on the Logical Memory II subscale  from the Wechsler Memory Scale-Revised (maximum score = 25) with (a) <8 for 16 years or more of education, (b) <4 for 8-15 years of education, (c) <2 for 0-7 years of education. The median MMSE score for the MCI patients was 25.3 (±1.4).The study was approved by the institutional review boards of Charité - Universitätsmedizin Berlin (EA1/182/10) and the study was performed in accordance with the Helsinki declaration. Written informed consent was obtained from all patients participating in the study.Samples and clinical data supplied by PrecisionMed are handled in strictest compliance with all applicable rules and regulations including the recommendations of the Council of the Human Genome Organization (HUGO) Ethical, Legal, and Social Issues Committee ; with the United Nations Educational, Scientific, and Cultural Organization's (UNESCO) Universal Declaration on the Human Genome and Human Rights (1997); and with recommendations guiding physicians in biomedical research involving human subjects adopted by the 18th World Medical Assembly, Helsinki, Finland, 1964 and later revisions.Total RNA including miRNA was isolated using the PAXgene Blood miRNA Kit (Qiagen) following the manufacturer's recommendations. Isolated RNA was stored at -80°C until use. RNA integrity was analyzed using Bioanalyzer 2100 (Agilent) and concentration and purity were measured using NanoDrop 2000 (Thermo Scientific).n = 48) and healthy controls (n = 22) by NGS.We first analyzed samples from AD patients . Preparation was performed following the protocol of the TruSeq Small RNA Sample Prep Kit (Illumina). Concentration of the ready prepped libraries was measured on the Bioanalyzer using the DNA 1000 Chip. Libraries were then pooled in batches of six samples in equal amounts and clustered with a concentration of 9 pmol in one lane each of a single read flowcell using the cBot (Illumina). Sequencing of 50 cycles was performed on a HiSeq 2000 (Illumina). Demultiplexing of the raw sequencing data and generation of the fastq files was done using CASAVA v.1.8.2.The raw Illumina reads were first preprocessed by cutting the 3' adapter sequence using the program fastx_clipper from the FASTX-Toolkit . Reads sPvalue) using Wilcoxon-Mann-Whitney (WMW) test. All significance values were adjusted for multiple testing using the Benjamini-Hochberg approach [For the NGS analysis, we excluded miRNAs with <50 read counts summed up across all samples of each group (AD or control), since these were considered lowly abundant. We normalized the read counts using standard quantile normalization. Next, we calculated for each miRNA the area under the receiver operator characteristic curve (AUC), the fold-change, and the significance value  [MiRNA enrichment analysis was performed using the TAM tool ,24. The te (FDR)  and werete (FDR) ,72).For validation purposes we analyzed the expression of single miRNAs using quantitative real time-polymerase chain reaction (RT-qPCR) in the same samples as used for NGS, if sufficient amounts of RNA were available. We used the miScript PCR System (Qiagen) for reverse transcription and RT-qPCR. A total of 200 ng RNA was converted into cDNA using the miScript Reverse Transcription Kit according to the manufacturer's protocol. The RT-qPCR was performed with the miScript SYBR® Green PCR Kit in a total volume of 20 μL per reaction containing 1 μL cDNA according to the manufacturer's protocol. For each miScript Primer Assay we additionally prepared a PCR negative-control with water instead of cDNA (non-template control).We further expanded the number of samples by further samples from patients with AD, MCI, CIS, PD, DEP, BD, and SCHIZ, resulting in a total of 202 samples analyzed by RT-qPCR .Click here for fileFigure S1. Heatmap for the 180 miRNAs significantly dys-regulated in AD patients compared to control individuals.Click here for fileTable S2. Table listing all novel mature miRNAs.Click here for fileTable S3. Table listing patient characteristics and indicates which samples are included in NGS analysis and/or in the RT-qPCR.Click here for file


This study aimed to investigate the effect of statins for the treatment of depression in individuals with acute coronary syndrome (ACS). We used 1-year follow-up data of a 24-week double-blind, placebo-controlled trial of escitalopram and a naturalistic prospective observational cohort study. Of 446 participants with comorbid depressive disorders and ACS at baseline, 300 participated in a randomised escitalopram trial and the remaining 146 participated in a naturalistic observational study. The participants in the two studies were approached for a 1-year follow-up investigation. Treatment response rates, defined as a ⩾50% reduction in the Hamilton Depression Rating Scale (HAM-D) and Beck Depression Inventory (BDI) scores, were used as the outcome variables. In the escitalopram trial, both HAM-D and BDI response rates were highest in patients taking escitalopram and statins together and lowest in patients receiving neither medication. Logistic regression analyses revealed that statin use was significantly associated with higher response rates on both the HAM-D and BDI at 1 year, whereas no such associations were found for escitalopram. In the naturalistic observational study, the response rates at 1 year did not differ significantly by statin use. Instead, the HAM-D response rate was significantly higher in patients taking lipophilic statins than in those who did not. In conclusion, statins may be effective for the treatment of depression independent of medical status and escitalopram use, and they may potentiate the antidepressant action of serotonergic antidepressants in patients with ACS. Furthermore, a few studies suggested that statins have an antidepressant effect5 independent of cholesterol-lowering effects.6 To date, only two small randomised controlled trials of statins has been published, suggesting antidepressant properties adjunctive to serotonin reuptake inhibitors in the general population with major depressive disorder.8Statins (3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitors) are widely used for primary and secondary prevention of cardiovascular disease by treating hypercholesterolaemia. Low mood and suicide have been reported as possible side effects of cholesterol-lowering treatment with statins.10 and statins have known effects on inflammatory cytokines, and also reduce oxidative stress markers.11 Statins exert anti-inflammatory activity, including reducing C-reactive protein concentration in both healthy individuals and those with stable vascular disease.12 In patients who experience an acute cardiac event, statins induce a rapid reduction in tumour necrosis factor alpha and interferon gamma production in stimulated T-lymphocytes, and inhibit the T helper cell (Th-1) immune response.13 In human hepatocytes, statins reduce interleukin-6-induced expression of C-reactive protein, suggesting a hepatic source of its anti-inflammatory effects.14 Furthermore, the low-density lipoprotein cholesterol-lowering effect of statins may be associated with anti-inflammatory effects, because low-density lipoprotein cholesterol itself strongly promotes inflammation.15 These anti-inflammatory effects and antioxidant effects of statins are thought to be associated with antidepressant effects.16The inflammatory hypothesis of depression is now well established,17 and a naturalistic prospective observational cohort study .18However, studies demonstrating the effects of statins on depression in individuals with acute coronary syndrome  are limited. In addition, to our knowledge, no previous reported study exists that identifies the interaction between statins and serotonin reuptake inhibitors on antidepressant effects in individuals with ACS. This study aimed to investigate the effects of statins on depression in a Korean population with ACS. We used 1 year follow-up data of a 24-week double-blind, placebo-controlled trial of escitalopram for treatment of depressive disorder in patients with ACS 18 Participants were recruited from those patients 2–14 weeks after an ACS episode that had been confirmed by various examinations and that resulted in hospitalisation (n=4809) in the central coordinating centre of the Korea Acute Myocardial Infarction Registry.The present study included subjects with comorbid ACS and depression at baseline in the K-DEPACS study. The K-DEPACS study is a naturalistic prospective cohort study that began in 2006. Comprehensive study details have been published previously.n=1152) were screened for depressive symptoms using the Beck Depression Inventory (BDI).19 Those with a score indicative of depression (>10) underwent a structured diagnostic evaluation conducted by research psychiatrists using the Mini-International Neuropsychiatric Interview,20 which categorises patients as having depressive disorder. Of them, 446 (38.7%) who met the Diagnostic and Statistical Manual of Mental Disorders, Fourth Edition (DSM-IV)21 criteria for major or minor depressive disorder were subjects in the present study. Of these, 300 (67.3%) participated in the EsDEPACS trial, which was a 24-week, double-blind randomised controlled trial of escitalopram . The remaining 146 individuals with depression did not participate in the clinical trial and only received standard medical treatment (K-DEPACS cohort). All participants in the two studies were approached for 1-year follow-up investigation. Written informed consent was obtained from all participants. Both studies were approved by the Chonnam National University Hospital Institutional Review Board.Patients who met the eligibility criteria and agreed to participate (n=149) or placebo (n=151). Statins were prescribed according to medical indications as determined by the treating cardiologists (n=226). To identify the effects of statins and interactions between statins and escitalopram, the 300 participants of the EsDEPACS trial were divided into four groups according to escitalopram and statin use, as follows: use of both escitalopram and statins , Escitalopram only , Statin only  and No medication  (P=0.225).In total, 300 patients from the EsDEPACS trial were randomised to either escitalopram  . A totalP=0.759).Among 146 participants who had depression in the K-DEPACS cohort study, 97 (66.4%) patients received a statin (statin group) and 49 (33.6%) patients received standard medical treatment only (no-medication group). A total of 111 subjects (76.0%) completed a 1-year follow-up evaluation, with no significant differences in follow-up rates among the two groups .−1 or if patients had a history of ongoing hyperlipidemia treatment. Body mass index was calculated, and participants were categorised as obese in cases of body mass index >25 kg/m2. To evaluate current cardiac status, diagnoses of myocardial infarction or unstable angina were made. ACS severity was estimated (range: I–IV) using the Killip classification.23 Left ventricular ejection fraction was estimated using echocardiography. Serum troponin I was measured as a cardiac biomarker.Various cardiac risk factors and medical status were investigated. Fasting total cholesterol level was assayed; hypercholesterolaemia was diagnosed when the cholesterol level was >200 mg dl17 In brief, subjects were randomised to the escitalopram and placebo conditions in a 1:1 ratio following computer-generated randomisation codes. Flexible doses of study medications ranging from 5 to 20 mg were administered for 24 weeks by a blinded psychiatrist. The mean (s.d.) doses at the last visit were 7.6 (3.7) mg for the escitalopram group and 8.5 (3.9) mg for the placebo group, and the mean (s.d.) treatment durations were 19.8 (3.2) and 19.7 (3.3) weeks, respectively.The details of the EsDEPACS trial have been published, in which superior effects of escitalopram to placebo were found.Concomitant medications, such as any other antidepressant, psychostimulant, antipsychotic or anticholinergic agents, were not permitted. In the K-DEPACS cohort, standard medical treatments were provided to all subjects by the study cardiologists based on international guidelines for the management of ACS. Outcome measures were blindly assessed following the prescription of statins as well as escitalopram.χ2-tests, analysis of variance or independent t-tests. Treatment response rates at 1 year, defined as a ⩾50% reduction in the HAM-D or BDI baseline score, were used as primary outcome variables. Response rates were also calculated at 24 weeks in the EsDEPACS trial. Response rates among the groups were compared using χ2-tests. Proportions of participants who no longer met DSM-IV criteria for major or minor depressive disorder at 1 year among the groups were also compared using χ2-tests. Repeated-measures analysis of variance treating time as the within-subject factor and treatment group as the between-subjects factor demonstrated changes in HAM-D and BDI scores over time in the EsDEPACS trial after adjusting for baseline scores. Logistic regression models were used to investigate independent effects of escitalopram and statins on treatment response after adjusting for baseline score of the depression measure, demographic and clinical characteristics that were not equally distributed among the groups (P<0.1) in unadjusted analyses, key cardiac parameters  and for each drug . In the EsDEPACS trial, 41 subjects randomly assigned to the escitalopram group discontinued the trial immediately after their baseline visit. Therefore, exploratory regression analyses of the 1-year follow-up evaluations excluded these subjects, who may have shown no medication effects. Another exploratory analyses were conducted to compare differential effects according to statin type: lipophilic statins  versus hydrophilic statins (rosuvastatin and pravastatin).24 A P-value <0.05 (two tailed) was taken to indicate statistical significance, and statistical analyses were performed using the SPSS software .The main analyses were performed on an intent-to-treat basis, including all patients who were randomly assigned to treatment. Demographic and clinical characteristics at baseline were compared according to treatment condition using P=0.091 and 0.090, respectively). In the K-DEPACS cohort, serum levels of troponin-I and total cholesterol were significantly higher in the statin-only group than in the no-medication group.Baseline characteristics by treatment status are compared in χ2=6.691, P=0.082).In the EsDEPACS trial, HAM-D and BDI response rates at both 24 weeks and 1 year differed significantly among treatment groups . Both HAIn the logistic regression, statin use was significantly associated with higher response rates on both the HAM-D and BDI at 1 year, whereas escitalopram use was not associated with the response rates on the HAM-D and BDI at 1 year after adjusting for significant covariates . HoweverP=0.010 and 0.030, respectively). Similar to the response-rate analyses, HAM-D and BDI scores were most improved in patients taking escitalopram and statins together.Escitalopram as a study medication was not provided to subjects after the 24-week trial period. Escitalopram was being taken by eight patients at the 1-year follow-up point on the basis of clinician's judgment. However, antidepressant use at 1 year did not differ significantly among the groups. Furthermore, the results were not changed substantially when the same analyses were repeated after excluding these participants.n=141) and hydrophilic statins (n=85), as the former theoretically have greater brain permeability. In the EsDEPACS trial, there was no significant difference in HAM-D or BDI response rates between these two groups .Exploratory analyses were conducted to compare the efficacy of lipophilic statins . However, there were no significant differences with respect to any other adverse events according to use of escitalopram or statin.Safety results were reported in our previous study.χ2=0.535, P=0.464). Logistic regression analysis also demonstrated no significant association between statin use with HAM-D or BDI response rates (HAM-D and BDI response rates at 1 year did not differ significantly according to statin use . Proportse rates . Antidepn=52) than in those who took hydrophilic statins (n=45) . No demographic or clinical characteristic differed significantly between the lipophilic and hydrophilic statins groups. Patients receiving hydrophilic statins were more likely to have diabetes . Logistic regression analysis was conducted to investigate the independent effects of lipophilic statins compared with hydrophilic statins after adjusting for baseline score of depression measure and clinical status  (P=0.054). When subjects were divided into two groups according to use of lipophilic statins, patients who took lipophilic statins (n=52) were significantly more likely to achieve HAM-D response at 1 year than those who did not .In the K-DEPACS cohort, HAM-D and BDI response rates were significantly higher in patients who took lipophilic statins (ication) . PatientAlthough there are a few studies suggesting beneficial effects of statins on depression in the general population, studies demonstrating antidepressant effect of statins in individuals with ACS are limited. The present study showed improvement in depressive symptoms associated with statin use in individuals with ACS and depressive disorder. These effects of statins at the 1-year follow-up point were independent of medical status and escitalopram use. However, in both subjective and objective measures, response rates in the group receiving combined statin and escitalopram treatment were highest among the four groups. To our knowledge, this is the first reported study to demonstrate an antidepressant effect of statins and an interaction between statins and escitalopram in individuals with ACS and depression.25 Inflammation, oxidative stress and stress reactions have also recently been suggested operative neurobiological pathways in depression.26 A growing body of literature illustrates the presence of increased inflammatory biomarkers, including inflammatory cytokines, in patients with depression.27 However, medications mainly targeting inflammatory and oxidative pathways have not been widely used in clinical settings. Our results are in accordance with previous novel clinical trials suggesting antidepressant effects of anti-inflammatory agents such as celecoxib, pioglitazone and N-acetylcysteine.31 Furthermore, findings of the highest response rate in the combination of escitalopram and statin group  suggests that statins may potentiate the efficacy of serotonergic antidepressants. This result is in accordance with a few recent controlled trials showing that augmentation of statins (lovastatin and atorvastatin) to serotonin reuptake inhibitors  may be effective for treating depression in the general population with major depressive disorder.8The pathophysiology and aetiology of depression are heterogeneous and complex. Monoamine deficiency was hypothesised to be the main cause of depression, and medications to compensate for this deficiency have been most widely used for the treatment of depression. However, the monoaminergic theory of depression has failed to deliver novel agents beyond the limited treatment options currently available.33 In this case, statins may be a novel alternative to treat depression or maintain the antidepressant effects of a serotonin reuptake inhibitor.In the intention-to-treat population of this study, the effect of escitalopram treatment on depressive symptoms was not observed at the 1-year follow-up point. This might be attributed to high proportions of subjects who had discontinued clinical trial after baseline visit. In fact, effect of escitalopram was seen at 1-year follow-up when the analysis was conducted in subjects who received escitalopram for at least 4 weeks. Given that almost all subjects had discontinued escitalopram at 24 weeks when the clinical trial was completed, and given that the mean duration of escitalopram treatment was about 20 weeks in the EsDEPACS trial, it appears that the effects of escitalopram use persist over a long period of time in patients with ACS. Our findings also suggest potential maintenance effects of the statin on depression. In some populations with ACS, escitalopram use may be limited due to its potential to increase the risk of bleeding or QTc interval prolongation.34 In addition, the clinical trial sample regularly met a psychiatrist for 24 weeks but subjects in the naturalistic observational study did not.In contrast to the results of clinical trial sample, statin use was not effective for the treatment of depression in the naturalistic observational study sample. This difference in result may be attributed to interactions of statin use with escitalopram as well as the placebo, which can biologically modulate the release of opioids, dopamine, serotonin and immune mediators.35 as is physical inactivity.36 These factors drive statin prescription, together with other risk factors for cardiovascular disease such as smoking, which itself is a risk factor for depression.37 As a consequence, one could reasonably expect participants prescribed statins to actually be at a considerably higher risk of depression. As a consequence, it is entirely feasible that the null finding in the non-randomised study, at least in part, reflects these factors. In addition, because of this residual confounding, non-randomised trials are likely to underestimate the antidepressant effects of statins.In addition, residual confounding in non-randomised trials is an important factor. The factors that drive statin prescription are known risk factors for depression. These data, as well as multiple other studies, suggest that obesity is a risk factor for depression. There is also extensive data that a poor-quality diet is a risk factor for depression,40 In the naturalistic observational study sample, the response rates with hydrophilic statins were significantly lower than those of lipophilic statins, which were similar to those with statins in the clinical trial sample. The relatively high response rates with hydrophilic statins used in the clinical trial sample, which resulted in the absence of a difference in effects according to the type of statins, may be also attributable to interactions with escitalopram and the placebo effect. The reason for the greater effect of lipophilic statins is unclear. The degree of lipophilicity required to pass the blood–brain barrier may be associated with direct effects on the brain. Specifically, lipophilic statins are more likely to cross the blood–brain barrier than are hydrophilic statins.41 In addition, lipophilic statins enter cells by passive diffusion and are thus widely distributed in different tissues, whereas hydrophilic statins are more liver-specific and are taken up via carrier-mediated mechanisms, thus reducing their ability to exert non-lipid effects in extrahepatic tissues.41There are two distinct types of statin, when classified by their lipophilicity. Lipophilic statins and hydrophilic statins exert differential effects on the human body.This study has some limitations. First, we should be cautious of generalising the antidepressant effects of statins to the general population, because this study was conducted only in individuals with ACS. Second, psychological treatment effects were not fully controlled in the clinical trial when patients met a psychiatrist who prescribed study medications. Third, the severity of our participants' depression was relatively low compared with that of patients in other clinical trials of depression; further study is warranted to investigate whether statins are effective in populations with severe depression. Fourth, the escitalopram-only group contained a relatively small number of subjects compared with the other groups. Furthermore, the study design combined a randomised controlled trial with a naturalistic observational study. Therefore, statins were administered to the subjects by cardiologists without randomisation, whereas administration of escitalopram was randomised; as mentioned above, this residual confounding may underestimate the effects of statins. However, depression assessments were conducted blind, independent of statin use. Further study should include randomisation of both escitalopram and statin to improve statistical power and remove residual confounding.In conclusion, the results of this study suggest that statins may be effective for the treatment of depression independent of medical status and escitalopram use, and they may potentiate the antidepressant action of serotonergic antidepressants in patients with ACS. In some patients with ACS and depressive disorder who are not tolerant to antidepressants, statins may be a novel alternative.


In 2014 the Joint United Nations Programme on HIV/AIDS defined the ambitious 90–90–90 targets for 2020, in which 90% of people living with HIV must be diagnosed, 90% of those diagnosed should be on sustained therapy and 90% of those on therapy should have an undetectable viral load. Children are considered to be a key focus population for these targets. This review will highlight key components of the epidemiology, prevention and treatment of tuberculosis (TB) in HIV-infected children in the era of increasing access to antiretroviral therapy (ART) and their relation to the 90–90–90 targets.The majority of HIV-infected children live in countries with a high burden of TB. In settings with a high burden of both diseases such as in sub-Saharan Africa, up to 57% of children diagnosed with and treated for TB are HIV-infected. TB results in substantial morbidity and mortality in HIV-infected children, so preventing TB and optimizing its treatment in HIV-infected children will be important to ensuring good long-term outcomes. Prevention of TB can be achieved by increasing access to ART to both children and adults, and appropriate provision of isoniazid preventative therapy. Co-treatment of HIV and TB is complicated by drug-drug interactions particularly due to the use of rifampicin; these may compromise virologic outcomes if appropriate corrective actions are not taken. There remain substantial operational challenges, and improved integration of paediatric TB and HIV services, including with antenatal and routine under-five care, is an important priority.TB may be an important barrier to achievement of the 90–90–90 targets, but specific attention to TB care in HIV-infected children may provide important opportunities to enhance the care of both TB and HIV in children. An estimated 240,000 children were newly infected with HIV in 2013 despite scale-up of prevention of mother-to-child transmission (PMTCT), which resulted in a 40% reduction in perinatal transmission of HIV in 2011 compared to 2009 [Tuberculosis (TB) remains a major cause of disease globally, with 9 million incident cases; 13% of these cases were HIV-infected. There were 1.5 million TB deaths in 2013, of which 360,000 were in HIV-infected persons. Although Asian countries (particularly China and India) account for the highest numbers of TB cases, all the countries with a TB incidence of more than 500 cases per 100,000 population are in Africa . In settGiven the close overlap of the TB and HIV epidemics, successful achievement of the 90–90–90 targets must specifically consider TB and its impact on HIV diagnosis, retention in care and attaining virologic suppression.In this paper we discuss key aspects of the care of TB in HIV-infected children, highlighting threats and opportunities on the road to 90–90–90. We will discuss the epidemiology of TB-HIV co-infection, calling attention to the ongoing close interactions of these two diseases, as well as recent advances in the prevention of TB in HIV-infected children. We will describe developments in the treatment of TB and HIV and the implications of these for the reduction of morbidity and mortality in TB-HIV co-infected children. We will also focus on the emerging threat of drug-resistant TB.As described above, the TB and HIV epidemics have substantial overlap in their epidemiology. It is clear that Africa (particularly sub-Saharan Africa), South East Asia (Thailand and India) and Latin America carry the biggest burden of TB and HIV ,6. This HIV-infected and HIV-exposed infants are highly TB exposed in their households and local communities. In a South African trial of isoniazid preventive therapy (IPT), 10% of HIV-exposed infants had contact with a potential case of TB by 14 weeks of age .In addition to frequent TB exposure, other conditions, including advanced immune suppression, poor nutritional status  and anaemia are associated with increased risk of TB in HIV-infected children . The proAmong South African infants with limited access to ART, up to 1595 cases per 100,000 population have been reported; this is more than 24 times the rate reported at the same time for HIV-uninfected South African infants . The risIn low-burden settings, the risk of TB in HIV-infected children is much less; however TB still occurs. Among children attending HIV services in the United Kingdom (between 1991 and 2006) and New York City (between 1989 and 1995) 3 to 5.5% had a diagnosis of TB at some point .TB co-infection negatively affects outcomes for HIV-infected patients, and there are challenges with TB diagnosis and co-treatment in HIV-infected children. These factors may all impact negatively on achieving 90% retention on ART and 90% virologic suppression; hence strategies to prevent TB in HIV-infected children are a priority for achieving the 90–90–90 targets.Increasing access to ART in paediatric cohorts decreases confirmed and probable TB. In the Children with HIV Early Antiretroviral Therapy (CHER) study, South African infants with deferred ART had 20 cases of TB per 100 person-years, compared to 8.3 cases per 100 person years in children receiving early ART . Up to 3The first three months after initiation of ART represent a very high-risk period for hospitalization and death . In addiWith increasing access to ART in adults, the risk of TB declines in adults as well as in HIV-infected and -uninfected children. In Johannesburg, South Africa, ART access in adults increased from 21.5% in 2005 to 68.2% in 2009 of those requiring ART. During the same period, TB in HIV-infected children declined from 1566.3 to 460.7 per 100,000 and in HIV-uninfected children from 18.7 to 11.0 per 100,000 . If ART Despite these clear benefits of ART, the risk of TB remains high in HIV-infected infants and children even when on ART. In South African infants with a 98% uptake of ART, those that were HIV-infected had a burden of 121 cases per 1000 years compared to 41 cases per 1000 child-years in HIV-exposed uninfected infants . This hiMycobacterium bovis strain, is protective against disseminated TB, including meningitis in young children, it is less effective at preventing pulmonary TB [Although Bacille Calmette Guérin (BCG), a live attenuated onary TB . BCG itsonary TB . In addionary TB . Given tonary TB . As BCG onary TB . In a pronary TB . Rather onary TB .Mycobacterium tuberculosis infection with isoniazid (INH) prevents disease [Young age is an important risk factor for the development of TB disease after infection . Treatin disease ,45. Ther disease .Studies of pre-exposure and longer-term IPT in HIV-infected children have yielded conflicting evidence. In the only true pre-exposure prevention trial (IMPAACT P1041), 548 HIV-infected and 804 HIV-exposed uninfected infants between three and four months of age without TB exposure were randomized to INH versus placebo for 96 weeks with follow-up for another 96 weeks. The pre-exposure prevention did not reduce the TB incidence in either HIV-exposed-uninfected or HIV-infected infants . An earlThere are substantial challenges in IPT implementation, including health system weakness and lack of availability of child-friendly INH formulations. Missed opportunities for chemoprophylaxis are well documented. A community-based study from South Africa found that only 21% of eligible children received IPT and that the lack of specific tools to ensure implementation possibly contributed to low uptake . Poor adIn HIV-uninfected children, adherence is improved by using rifamycins, which require shorter courses . This isThere is no published data in HIV-infected children on the benefit of tuberculin skin testing or other tests of infection, such as the interferon gamma release assays, to help target pre-exposure IPT. Data in HIV-infected adults suggests that TST-positive adults benefit the most from IPT . No TB pThere are a number of other additional opportunities for preventing TB in HIV-infected children. Temporal associations between TB, influenza and pneumococcal disease in children suggest a complex interaction between these infections. An increase in TB diagnoses following approximately three months after an influenza epidemic in a high burden setting suggests a potential role for the influenza vaccine in reducing the TB burden . A studyPrior to wide access to ART, high mortality of co-infected children was reported. A study from South Africa documented a 10 times higher mortality in TB-HIV co-infected children than in HIV-uninfected children with TB . ChildreConcomitant TB therapy may be a significant risk factor for virologic failure in children who are co-treated, impacting the target of 90% of children on ART being virologically suppressed. Viral load is not available in all settings where there is a high TB burden. In settings where the protease inhibitor (PI) lopinavir/ritonavir (4:1) LPV/r is the preferred initial therapy in children<3 years and super-boosting is used, high levels of viral suppression are reported for children, regardless of co-treatment. However the rates of suppression at 6 and 12 months are lower than those of children not requiring co-treatment –64. StudNo difference in virologic suppression has been seen with efavirenz (EFV)-based therapies at month 12 . There iOptimizing the co-treatment of TB and HIV is important to ensuring the targeted 90% treatment success in HIV-infected children.In low resource settings, TB treatment initiation is as frequent a cause of ART regimen changes as adverse effects . When chIn 2010, the WHO recommended higher doses of the first-line anti-TB drugs in all children . This ren=5) had a significantly lower Cmax and Tmax on PZA 35 mg/kg, but no difference in total exposure; the small sample size may have limited the ability to detect other differences [n=7 children) was associated with a low two-hour INH concentration [Among 20 children younger than two years receiving the newly recommended doses of INH, RMP and PZA, nearly all children had maximum serum concentrations of these medications above target levels. HIV-infected children in this study , PI and integrase inhibitors. For some of these interactions, there is no paediatric data.max and AUC0–12 were lower than in controls, but the Cmin (the target for efficacy) was greater than the minimum recommended. Children tolerated the strategy and two cases had mild alanine transaminase elevation that did not require therapy interruption [LPV/r is superior to NVP in young children regardless of previous NVP or EFV exposure through PMTCT ,90. LPV/rruption . Prelimirruption .2 twice), 80% did not achieve a target morning trough of 1 mg/L [In adults, giving double the dose of LPV/r was found to have acceptable pharmacokinetic and less toxicity . Howeverf 1 mg/L . Explanaf 1 mg/L . The indRifabutin, which does not affect PI concentrations, is also problematic. Rifabutin is metabolized by CYP3A4 and dose adjustments of rifabutin are needed if co-treating with PIs; however pharmacokinetic data in children is limited. In six children treated with rifabutin 5 mg/kg three times per week and with LPV/r, severe transient neutropenia and insufficient rifabutin exposure was observed . Using rThere is a lack of paediatric data for boosted atazanavir (ATV) and darunavir (DRV), which also both interact with RMP. In adult volunteers, double doses of ATV with RMP did not correct the ATV exposure but did not cause substantial toxicity . For DRVEFV is used in older children and is superior to NVP . Recent 2/dose twice daily should be used [NVP is a commonly used NNRTI in low resource settings, where it is incorporated in easy-to-use and well-tolerated FDCs. There are reports of adequate exposure in RMP co-treatment but larger co-treatment studies found significant under-dosing even if NVP was given at more than the standard recommended dose –106. The be used . Switchi be used . These lSwitching suppressed children from LPV/r to EFV is a strategy studied in children without TB . AlthougEtravirine is not typically available in low resource settings and is not approved for use in children less than six years of age. There are no adult studies assessing the interaction of RMP and etravirine, with the exception of case reports, which confirm a significant reduction in plasma levels, which did not impact virologic suppression .Based on results of the Antiretroviral Research for Watoto (ARROW) study, the WHO recommends triple NRTIs as a preferred strategy in children age <3 years requiring RMP co-treatment. In ARROW, three NRTIs were studied as an intensive induction and a maintenance strategy when combined with NNRTIs. After 48 weeks of initial therapy that contained a NNRTI children were randomized to a three-drug NRTI maintenance regimen or to remain on conventional therapy. Fifty-nine of the 1143 children required a drug alteration for TB that included stopping or replacing NVP. Children on EFV did not require drug switch. Triple NRTI was effective at 36 weeks, but not at 144 weeks. The advantage of this strategy is that, although viral suppression is inferior, the risk of progressive NNRTI and PI resistance was avoided and complicated drug-drug interactions and logistic complications were easier to manage . Where PIntegrase inhibitors are an increasingly important class of ART, particularly as a key component of third-line regimens. Raltegravir and dolutegravir are not metabolized by the CYP P450 enzymes but are metabolized through the liver by uridine 5-diphospho (UDP)-glucuro-nosyltransferase 1A1, an enzyme that is also induced by RMP. Healthy volunteer studies showed a significant reduction in raltegravir exposure with RMP co-treatment . In a prAdult healthy volunteer studies assessing the effect of RMP on dolutegravir suggest that doubling the dolutegravir dose is needed if there is co-treatment with RMP .Children needing third-line therapy and co-treatment with RMP-containing TB therapy may benefit from a holding triple NRTI strategy. This may be a good option if the children have a preserved CD4 count and are clinically stable. Where children require a suppressive regimen urgently, consideration should be given to changing to a non-rifamycin-containing TB regimen. Rifabutin also has less substantial interactions, but dose adjustment of rifabutin and ART may still be needed. Rifapentine is not recommended, as there may be a risk of RMP-monoresistant TB developing . FluoroqThe timing of ART initiation in adults with TB has been extensively studied. Delaying therapy in adults with a CD4 count of <200 cells/mL has been associated with poor virologic and clinical outcomes . The timTB meningitis (TBM) is an exception regardless of CD4 count, since clinical deterioration due to paradoxical IRIS can be devastating. In the adult literature, IRIS is associated with more disseminated forms of the disease and positive culture in the cerebrospinal fluid. It is common practice to delay ART four to six weeks in TBM . AlthougIf children are already on ART, appropriate anti-TB therapy should be started as soon as the appropriate diagnostic testing has been performed. ART should be adapted and the possibility of virologic and or immunological failure must be considered and appropriately investigated.MDR-TB (resistant to INH and RMP) is a growing health threat with an estimated 480,000 cases occurring in 2013; 9% of these cases also had additional resistance to a fluoroquinolone and/or a second-line injectable medication –120. HIVInitially TB and HIV services were introduced as vertical programmes, but now strategies to integrate these services and strengthen the linkage to antenatal care are essential to improving diagnosis and access to care and treatment. HIV testing remains the entry point for HIV care. Ensuring that children diagnosed with TB are tested for HIV is absolutely essential. Given the high risk of HIV co-infection in children with TB in many settings, routine HIV testing of all children receiving TB treatment is an important opportunity to increase HIV diagnosis and thus achievement of 90% of HIV-infected persons knowing their status.The diagnosis of TB in all children remains challenging. A study of paediatric ART programmes in diverse resource-limited settings showed that, although sputum microscopy and CXR were available in all programmes, they were only used in 86 and 52% of TB diagnoses. Xpert MTB/RIF was only used in 8% of TB diagnoses and mycobacterial culture in 17%. Eighty-six percent of sites provided access to TB treatment, but 30% never provided IPT to children . Adult cScreening of pregnant women for both HIV and TB may improve access not only to PMTCT, but also to IPT. Integrating TB services into antenatal care of HIV-infected women may also be key to preventing TB in young infants . In TablThe TB and HIV epidemics remain closely interlinked and TB is still an important opportunistic infection in HIV-infected children, with substantial mortality and morbidity and with co-treatment possibly affecting HIV outcomes. The growing burden of drug-resistant TB poses new challenges. Increasing ART access has the potential to greatly impact the TB epidemic in settings with high dual burden. The reduction in adult cases will reduce TB infection to both HIV-infected and -uninfected infants and children. Early ART in HIV-infected children will further reduce the burden of TB in these children. The provision of IPT should be strengthened, and innovative prevention strategies such as influenza and pneumococcal vaccination and continuing co-trimoxazole should be explored. Treating these diseases simultaneously presents challenges with regards to choosing the most appropriate regimens and ensuring that medications are available in all settings and easy for children to adhere to. There is a synergy between working towards the 90–90–90 targets and improving TB diagnostic and treatment programmes. Efforts to meet the 90–90–90 targets for both adults and children may well have a profound impact on the burden of childhood TB, while improved prevention, diagnosis and treatment of TB in co-infected children, as well as strengthening and integration of TB-HIV programmes, will be important if the 90–90–90 targets are to be achieved.


The effects of olive oil phenolic compounds (PCs) on HDL proteome, with respect to new aspects of cardioprotective properties, are still unknown. The aim of this study was to assess the impact on the HDL protein cargo of the intake of virgin olive oil (VOO) and two functional VOOs, enriched with their own PCs (FVOO) or complemented with thyme PCs (FVOOT), in hypercholesterolemic subjects. Eligible volunteers were recruited from the IMIM-Hospital del Mar Medical Research Institute (Spain) from April 2012 to September 2012. Thirty-three hypercholesterolemic participants  were randomized in the double-blind, controlled, cross-over VOHF clinical trial. The subjects received for 3 weeks 25 mL/day of: VOO, FVOO, or FVOOT. Using a quantitative proteomics approach, 127 HDL-associated proteins were identified. Among these, 15 were commonly differently expressed after the three VOO interventions compared to baseline, with specific changes observed for each intervention. The 15 common proteins were mainly involved in the following pathways: LXR/RXR activation, acute phase response, and atherosclerosis. The three VOOs were well tolerated by all participants. Consumption of VOO, or phenol-enriched VOOs, has an impact on the HDL proteome in a cardioprotective mode by up-regulating proteins related to cholesterol homeostasis, protection against oxidation and blood coagulation while down-regulating proteins implicated in acute-phase response, lipid transport, and immune response. The common observed protein expression modifications after the three VOOs indicate a major matrix effect.ISRCTN77500181.International Standard Randomized Controlled Trials  To date, studies have been focused on hydroxytyrosol because it has been reported as being the most biologically active PC in OO [Olive oil (OO) is a food item typical of the Mediterranean diet, and several studies have revealed that it has a unique phenolic profile with specific biological properties. Results from the European EUROLIVE study  showed tPC in OO . In factPC in OO . As the PC in OO ,4. FurthPC in OO . MoreovePC in OO . For thePC in OO ,8.Approximately one hundred different proteins, not considered to be apolipoproteins (APO), have been identified as being associated with the HDL particle  and linkIn this context, we hypothesized that sustained consumption of PCs from VOO, or functional phenol-enriched VOO, could modify the HDL proteome, which in turn could be related to their cardioprotective benefits. The objective of the current study was to assess the impact on the HDL protein cargo of a dietary intervention supplemented with a VOO or two different functional VOOs enriched with their own PCs  or complemented with thyme PCs , in hypercholesterolemic subjects from the VOHF study.www.internationaloliveoil.org; ISRCTN77500181). This trial was conducted according to extended CONSORT 2010 guidelines.The VOHF study aimed at assessing whether functional VOOs, enriched with their own PCs or with them plus complementary phenols from thyme, could have a nutraceutical effect on HDL lipoprotein functionality. The present clinical trial was conducted in accordance with the Helsinki Declaration and the Good Clinical Practice for Trials on Medical Products in the European Community. All participants provided written informed consent, and the local institutional ethics committees approved the protocol  registered with the International Standard Randomized Controlled Trial register , diabetes, multiple allergies, intestinal diseases, or any other disease or condition that could worsen compliance. The study was conducted at IMIM- Hospital del Mar Medical Research Institute (Spain) from April 2012 to September 2012. The participants’ flow chart is described in The VOHF study was a double-blind, randomized, controlled, crossover clinical trial with 33 hypercholesterolemic volunteers , aged 35 to 80 year. Exclusion criteria included the following: BMI >35 Kg/mSubjects were randomly allocated to one of 3 sequences of administration of 25 mL/day of raw: a) VOO; 80 mg of PCs/kg oil, b) functional VOO enriched with its own PCs , and c) functional VOO enriched with its own PCs plus complementary phenols from thyme   . The ranThymus zyguis). The phenolic extracts used for enrichment were obtained in the laboratory using an accelerated solvent extractor . The procedure to obtain the phenolic extracts and the enriched oils has been previously described . BrieflyThe sample size of 30 individuals allows at least 80% power to detect a statistically significant difference among three groups of 3 mg/dL of HDL-C and a standard deviation of 1.9, using an ANOVA test and assuming a dropout rate of 15% and a Type I error of 0.05.2EDTA anticoagulant. Blood samples were centrifuged at 1 500 xg for 15 minutes and 2.8 mL of plasma were finally recovered. Protease Inhibitor Cocktail  was added to plasma at a concentration 1/100 (1 μL of PIC for 100 μL of plasma). All samples were stored at -80°C until processing. A total of 123 plasma samples were used for proteomic analyses (33 participants per 4 visits minus 9 missing). See proteomic study flow chart (Fasting blood samples were taken from 33 participants before the first washout period (first visit-baseline) and after each VOO intervention period , through a catheter in an antecubital vein . Blood wow chart .g for 21 h at 10°C after adjustment of the plasma density to 1.063mg/mL with NaBr. The HDL fraction was obtained by adjusting the infranatant density to 1.21 mg/mL with NaBr prior to ultracentrifugation at 140 000 xg for 40 h at 10°C. 2 mL of HDL fraction were recovered and later stored at -80°C.The HDL fraction (d = 1.036–1.21 g/mL) from each sample was isolated from 2.5 mL of plasma by sequential density ultracentrifugation in two steps using sodium bromide , as previously described by Havel RJ, et al. . SequentApo A-I, Apo A-II and Apo B-100 were determined in each volunteer HDL fraction using immuno-turbidimetry assays  in an autoanalyzer Cobas-Mira Plus . Total Cholesterol, Free Cholesterol, Phospholipids and Triglycerides were determined by enzymatic colorimetric techniques  also in the autoanalyzer Cobas-Mira Plus. Total protein was quantified in HDL fractions by the Bradford method . HDL fraction purity was assessed through the measurement of Apo B-100, almost all samples exhibited concentrations below the limit of detection (2 mg/dL).S1 Supporting Information.For the proteomics studies (detailed in Supporting Information Data) the 123 isolated HDL samples were reduced to ten pools: 1 from first visit- baseline, 3 after third visit, 3 after fifth visit, and 3 after seventh visit . The pooVarious bioinformatics tool were employed for the biological interpretation of the results. Proteins are referred to by their gene encode symbol.http://string-db-org). Ingenuity Pathway analysis  was used to analyze canonical pathways and protein networks involving the differentially expressed proteins for biological interpretation (p<0.01). The differentially expressed proteins were overlaid with IPA-curated canonical pathways to explore possible metabolic and cell signaling pathways that were over- or under-represented by the experimentally determined genes. Specifically, we analyzed the proteins that overlapped the three VOO interventions in order to investigate potential common OO PCs mechanisms. In addition, possible connections between mapped genes were evaluated and graphical networks were algorithmically generated. Nodes representing genes and gene products were linked by biological relationships. Networks were ranked by a score that defines the probability of a collection of nodes being equal to or greater than the number in a network achieved by chance alone.Hierarchical clustering of protein expression data to investigate overall similarities of the proteome samples and identify the main biological functions involved was performed in web-tool STRING 9.1  in an autoanalyzer Cobas-Mira Plus . The quantification of haptoglobin and clusterin was performed using two commercial ELISA assays following manufacter’s instructions; the AssayMax Haptoglobin ELISA kit  and the Human Clusterin ELISA Kit , respectively.p < 0.05. The possible interaction between the treatments and the treatment sequence (carryover effect) was assessed testing the period by treatment interaction-effect under a linear mixed model. All statistical analyses were performed with Statistical Package for the Social Sciences (SPSS) for Windows .The Kolmogorov-Smirnov test was used to verify the distributions of the variables. A paired Student’s t-test was employed for the comparison of paired and normally distributed variables. Wilcoxon signed-rank test was used for the comparison of paired and non-normally distributed variables. The level of statistical significance was set at Biochemical characterization of HDL and also Apo A-I and HDL-c plasma levels segregated according to VOO intervention sequence are described in detail in A total of 155 HDL-associated proteins were initially identified by applying shotgun proteomics using Maldi and Orbitrap MS/MS to HDL fractions isolated by ultracentrifugation. Data were subsequently screened to ensure protein identification accuracy and 28 possible false-positives removed. The final list of HDL-associated proteins identified both at baseline and after interventions, was refined to 127 proteins . The majCompared to baseline values (first visit), the HDL protein cargoes of the differentially expressed proteins differed after the interventions according to the VOO received. The complete list of the proteins differentially expressed after each VOO intervention, and their principal biological functions, are shown in In addition to these 15 commonly modulated proteins, other specific protein expression changes were observed after each OO intervention. The stronger effects for each VOO were related to the following biological functions linked to atherosclerosis protection: transport, homeostasis cholesterol, antioxidant protection, blood coagulation, innate immune response, acute phase, and cellular adhesion. A stronger protein expression change was observed after VOO intervention in the following 6 proteins: Apo C-I, Apo A-I, Cholesteryl ester transfer protein (CETP), PON3, histidine-rich glycoprotein (HRG), and Ig heavy chain V-I region HG3, which were up-regulated while Ig lambda chain V-III region LOI was down-regulated. A stronger effect after FVOO was observed on 2 protein expression changes, PON3 and platelet basic protein (PPBP) which were up-regulated. Finally, a stronger up-expression change was noticed after FVOOT in the following 7 proteins: Apo A-I, Apo A-IV, Apo C-II, Apo C-III, serum amyloid A protein 4 (SAA4), afamin (AFM) and integrin beta-3 (ITGB3).A panel of four proteins identified by MS analysis with changes after the three interventions, two up-regulated (Apo AI and Apo AII) and two down-regulated (Clusterin and Haptoglobin), were selected to confirm the changes in the protein expression that were observed in the proteomic analysis. These four proteins were quantified in the same 10 HDL pools used for proteomic analysis. Apo A-I and Apo A-II increases observed by proteomics were confirmed by immunoturbidimetry. The fold-changes observed in Apo A-I and Apo A-II measured by immunodetection were approximately 1.2 after the three VOO interventions compared to the basal value . Moreovep<0.01) by OO phenols. The top 7 signaling pathways modified by the differentially expressed common proteins after all VOO interventions included: LXR/RXR activation, acute phase response signaling, atherosclerosis signaling, IL-12 signaling and production in macrophages, production of nitric oxide (NO) and reactive oxygen species (ROS) in macrophages, Clathrin-mediated endocytosis signaling, and coagulation system , followed by acute phase response signaling . The highest scoring associated network generated by IPA, and the differentially expressed proteins common for all VOO interventions, are shown in p<0.01). NR5A2, which is implicated in the canonical pathway of FXR/RXR activation and the biological process of cholesterol homeostasis, was predicted from our results to be activated. Likewise, IL-6, which is involved in the acute phase response canonical pathway, was expected to be inhibited.IPA analysis was performed to reveal the canonical pathways significantly affected  of the common matrix of the three VOO tested on HDL remodeling. Moreover, IPA analysis revealed that several signaling pathways related to CVD were affected by OO PCs consumption. These data emphasize the key role of OO PCs in modifying pathways and conferring cardioprotective properties. PCs of VOO could be capable of transcriptional gene regulation inducing the down or up-regulation of proteins involved in functions related to cardiovascular risk ,19. FarrTo our knowledge, this is the first HDL proteomic study in humans that assesses the effects of PCs on the HDL protein cargo.Two well-known proteins related to cholesterol homeostasis, Apo A-I and Apo A-II, were significantly augmented after the three VOO interventions. Both proteins participate in reverse cholesterol transport and present anti-inflammatory and antioxidant properties ,25. The Two proteins related to blood coagulation, SERPIND1 and A2M, were up-regulated after the three VOO interventions thus increasing the cardioprotective activity of the HDL particle.PON3, which is an antioxidant enzyme associated to HDL and with lactonase activity , was up-AHSG, ORM1, SERPINF2, and HP are acute-phase response proteins which were down-regulated after all VOO interventions. Acute-phase proteins are increased during inflammation and could be used as inflammatory biomarkers related to CVD ,31. Our Plasma Apo D and RBP4, which have been reported to be up-regulated during CVD development ,34, decrCLU, or Apo J, has been suggested to play a compensatory protective role by acting as an inflammatory modulator in myocardial infarction . In the In addition to the commonly modulated proteins, specific strong effects on the HDL protein cargo were also observed after each intervention. For instance, after the intervention with VOO, Apo C-I, Apo A-I, CETP, and PON3 were markedly up-regulated. Among these changes the up-regulation of CETP is noteworthy. This protein facilitates the transport of cholesteryl ester from HDL to Apo B-100 containing lipoproteins, and its plasma activity has been suggested to be inhibited after a PC intervention . To our From all the marked expression changes observed after the FVOO intervention, we would like to emphasize the up-regulation of the PON3.We also highlight the strong up-regulation of Apo A-I, Apo A-IV, Apo C-II, and Apo C-III among the changes observed after the FVOOT intervention. Apo A-IV is mainly related to cholesterol homeostasis and oxidative protection, and has been associated with the cardioprotective properties of the HDL particle . AlthougThe pooling sample approach employed for the proteomic analysis, which was used to focalize primarily on the whole protein response and also to optimize the associated research expenses, constitutes the limitation of our study. Because of the methodological process used, the results of this work represent an exploratory analysis about the effect of a dietary intervention supplemented with a VOO or two different phenol enriched VOOs on the HDL proteome. However, the analysis is not sufficiently robust to draw conclusions about which of the three VOO tested has had the stronger effect on the HDL protein cargo. Future studies which analyze individual samples instead of pooling samples should be done to better assess the difference in the impact produced by the three types of VOO tested.Moreover, HDL isolation is a key methodological point for proteomic analysis of HDL. Currently, the gold standard method for the HDL isolation is still under discussion and future attempts will be targeted towards improving isolation procedures . Whilst One of the strengths of this study is its randomized and crossover design, which permitted the participants to ingest all VOO types. Moreover, the identification of the HDL-associated proteins was done by with two different MS techniques, Orbitrap and Maldi, which provide reliability and robustness to our identification results. Furthermore, it is important to note the novelty of the subject studied. The dietary modifications used in this study were very specific considering that only the concentration and the source of PCs of VOO administrated changed between interventions. The observed changes in the HDL proteome are smaller than those found when comparing a healthy population with patients with acute coronary syndrome  or with The results of our study illustrate the potential of HDL proteomics to lead to new biomarkers for CVD prevention. These HDL proteomics findings help to understand the improvement of the HDL functionality by measuring the effectiveness of nutritional interventions in a similar manner to pharmacological treatments as proposed by Birner-Gruenberger . In concS1 Checklist(DOC)Click here for additional data file.S1 Protocol(DOC)Click here for additional data file.S1 FigProteins were presented with their gene encode symbol. Nodes represent genes encoding interacting proteins and the lines between them represent known and predicted interactions.(TIFF)Click here for additional data file.S1 File(DOCX)Click here for additional data file.S1 Table(DOCX)Click here for additional data file.S2 TablePhenolic compounds, fat soluble micronutrients and fatty acids daily intake through 25 mL of VOO, FVOO and FVOOT.(DOCX)Click here for additional data file.S3 Table(DOCX)Click here for additional data file.S4 Table(DOCX)Click here for additional data file.S5 Table(DOCX)Click here for additional data file.