To determine whether there are differences in autonomic nervous system function in early- versus late-onset preeclampsia.+0 weeks of gestation (early-onset preeclampsia) and ≥ 34+0 weeks of gestation (late-onset preeclampsia). For each case in each of the preeclampsia subgroups, three „control”uncomplicated singleton pregnancies were matched by maternal age, height, and week of gestation. Blood pressure and heart rate were measured continuously for 30 minutes in each participant. Baroreceptor reflex sensitivity (assessed using sequence technique), time and frequency domain heart rate variability measures, as SDNN, RMSSD, LFRRI, HFRRI and LF/HFRRI of R-R intervals, were compared between groups (p<0.05 significant).Matched case-control study. Cases were defined as singleton pregnancies with preeclampsia at < 34RRI were significantly higher in the late-onset preeclampsia group compared to gestational age matched controls . No significant differences in SDNN RMSSD and HFRRI between early-onset preeclampsia group and gestational age matched controls were observed . Similarly, baroreceptor reflex sensitivity was higher in late-onset preeclampsia compared to controls at ≥ 34 weeks (p = 0.037), but not different between early-onset preeclampsia compared to controls at < 34 weeks (p = 0.50).24 women with preeclampsia (10 with early-onset and 14 with late-onset preeclampsia) and 72 controls were included in the study. SDNN, RMSSD and HFHeart rate variability and baroreceptor reflex sensitivity are increased in late- but not early-onset preeclampsia compared to healthy pregnancies. This indicates a better autonomic nervous system mediated adaptation to preeclampsia related cardiovascular changes in late-onset disease. Preeclampsia, a multisystem disorder characterized by new-onset hypertension and proteinuria or end-organ dysfunction after 20 weeks of gestation, affects 2 to 8% of pregnancies –7. It isThe autonomic nervous system plays a central role in cardiovascular adaptation to pregnancy-related hemodynamic changes –18. SeveThe aim of the study was to determine whether there are significant differences in autonomic nervous system function in pregnancies complicated by early- compared to late-onset preeclampsia and normal pregnancies.+0 weeks of gestation at the time of diagnosis (early-onset preeclampsia) and ≥ 34+0 weeks of gestation at the time of diagnosis  hospitalized at our tertiary perinatal center. For each case in each of the preeclampsia subgroups, three „control”singleton pregnancies without preeclampsia and pre-existing diseases  were matched by maternal age, height, and week of gestation.The study design utilized a matched case-control methodology. Cases were defined as singleton pregnancies with preeclampsia at < 34Gestational age was derived from the last menstrual period that was confirmed by first trimester ultrasound scans. Preeclampsia was defined using the American College of Obstetricians and Gynecologist Task Force on Hypertension in Pregnancy recommendations .After participants were familiarized with the test protocol, equipment and personnel, electrodes were attached and patients were positioned in the 15° left lateral position, ensuring a continuous venous blood flow to the heart. During the whole procedure the participants were asked not to talk or make abrupt movements. The study protocol consisted of an adaptation period of 20 min and 10 min recording at rest. For analysis a five minute epoch commencing at 4 min of rest was used.range = 50–250 mmHg, ±5 mmHg) and hearth rate  were carried out with the Task Force® Monitor  [Continuous monitoring of blood pressure  . ContinuAustria) .For the purpose of this study we analyzed time domain and frequency domain measures of heart rate variability. Time domain measures were computed as the standard deviation of normal-to-normal beat (SDNN) and root mean squared successive differences (RMSSD) of R-R intervals. SDNN reflects sympathetic and to some extent vagal tone whereas RMSSD reflects vagal tone only .nd order. Low frequency (LFRRI) was defined as 0.04–0.15 Hz, high frequency (HFRRI) was defined as 0.15–0.40 Hz, according to published recommendations and LF/HFRRI, an indicator of the autonomic state was computed [RRI), ln(HFRRI), ln(LF/HFRRI)).For frequency domain measures of the variability of R-R intervals (RRI) we used Fast Fourier Transform with a Hanning window for spectral analysis of cardiovascular signals on the blocks of 5 min epochs, after resampling and removing the trend of 2computed . BecauseThe sequence technique was used for the assessment of BRS ,26. ThisIn addition, further data which are not relevant for this research question were analyzed .Data are expressed as means ± standard deviations. ANOVA was used for comparison between the groups with early-onset preeclampsia vs. late-onset preeclampsia vs. controls. For all tests, a two-tailed p value ≤ 0.05 was considered statistically significant. The software used for statistical analysis was IBM SPSS Statistics for Windows Version 21.0 .The study was performed in accordance with the 1964 Declaration of Helsinki and was approved by the Ethics Committee of the Medical University of Graz. Written informed consent was obtained from all participants.mild , in the other cases as severe.Twenty-for women with preeclampsia (10 with early-onset and 14 with late-onset preeclampsia) and 72 controls were included in the study. In five women the severity of preeclampsia were classified as Demographic data for the 96 study participants are presented in There were no significant differences in the heart rate between preeclamptic patients and healthy controls overall as well as between the preeclampsia groups compared to gestational age matched controls .SDNN did not differ between preeclamptic patients and healthy controls overall (p = 0.132), whereas RMSSD was higher in preeclamptic patients (p = 0.027). Furthermore, SDNN and RMSSD were significantly higher in the late-onset preeclampsia group compared to gestational age matched controls . No significant differences in SDNN and RMSSD between the early-onset preeclampsia group and gestational age matched controls were observed .RRI), ln(HFRRI) and ln(LF/HFRRI) between preeclamptic patients and healthy controls overall . However, ln(HFRRI) was significantly higher in the late-onset preeclampsia group compared to gestational age matched controls (p = 0.018). No difference for this group were seen for ln(LFRRI) and ln(LF/HFRRI) . In a line with the time domain measures no significant differences in the frequency domain measures ln(LFRRI), ln(HFRRI) and ln(LF/HFRRI) between early-onset preeclampsia group and gestational age matched controls were observed .The ANOVAs yielded no significant differences regarding frequency domain measures ln. BRS was higher in late-onset preeclampsia compared to controls at ≥ 34 weeks (p<0.037), but not statistically different between early-onset preeclampsia compared to controls at < 34 weeks (p = 0.500).Our results show that late-onset preeclampsia, but not early-onset preeclampsia, is associated with increased heart rate variability and BRS compared to matched healthy pregnancies. The sequence technique used for the assessment of BRS provides an index of regulatory activity of the autonomic nervous system on the R-R intervals. The cardiac branch of the baroreflex, which regulates the interaction between blood pressure and R-R intervals, is one relevant source of parasympathetic influences in cardiac autonomic regulation , 28. HypFaber et al. showed that heart rate variability and BRS differed between different hypertensive disorders of pregnancy , suggesting that different manifestations of hypertension in pregnancy have different pathophysiological, regulatory, and compensatory mechanisms . HoweverIn our study no difference in BRS and HRV variables except RMSSD were observed between the 24 preeclamptic women and the 72 case-matched healthy controls. However, the separate analysis of early-onset and late-onset preeclamptic women compared to the matched healthy pregnancies showed a difference in BRS and HRV of late-onset compared to healthy pregnancies. Instead, no difference for early-onset compared to healthy pregnancies were observed supporting the findings of Faber et al. Differences between our findings and their study might also be explained by the small number of patients with early-onset preeclampsia in our study. Furthermore, in contrast to our results, previous studies demonstrated a physiological decrease in heart rate variability and BRS throughout normal pregnancy ,32. TherDespite the relatively small sample size, the study also has several strengths. It is the first study assessing changes in heart rate variability and BRS in early- and late-onset preeclampsia separately. Furthermore, the methods used for the assessment of heart rate variability and BRS are non-invasive and their validity has been verified in animal studies, numerous clinical studies outside pregnancy, as well as in pregnant women ,33–37.In conclusion, the results of our study suggest significant differences in autonomic nervous system responses in early- vs. late-onset preeclampsia. This suggests that these two preeclampsia subtypes should be managed as two distinct entities not only from an etiologic and prognostic standpoint, but also in terms of treatment options. Non-invasive measurements of BRS and heart rate variability could be of great clinical importance not only for prediction and diagnosis of preeclampsia, but also for management of patients with already established clinical signs of disease. As objective markers of autonomic nervous system function these measurements could help clinicians individualizing antihypertensive, volume and diuretic therapy which would lower the risks of side effects and additional end-organ damage in patients with preeclampsia.S1 TableDataset of the reported data plus blood pressure values.(XLS)Click here for additional data file.


Parents interact with children following specific styles, known to influence child development. These styles represent variations in the dimensions of demandingness and responsiveness, resulting in authoritarian, authoritative, permissive or uninvolved parenting. Given the similarities in the parent to child and owner to dog relationships, we determined the extent to which parenting styles exist in the owner to dog relationship using the existing Parenting Styles and Dimensions Questionnaire for the parent-child relationship and an adapted version for dog owners. Items on the parenting of children/dogs were rated for applicability on a five-point Likert scale by 518 Dutch dog owning parents. Principal Component Analyses grouped parenting propensities into styles, with some marked differences between the findings for children and dogs. Dog-directed items grouped into an authoritarian-correction orientated style, incorporating variation in demandingness and focussing on correcting a dog for behaviour verbally/physically, and in two styles based on authoritative items. An authoritative-intrinsic value orientated style reflected variation in mainly responsiveness and oriented on the assumed needs and emotions of the animal. A second authoritative-item based style, captured variations in demandingness and responsiveness. We labelled this style authoritative-training orientated, as it orientated on manners in teaching a dog how to behave in social situations. Thus, we defined dog-directed parenting styles and constructed a Dog-Directed Parenting Styles and Dimensions Questionnaire along the lines of the existing theoretical framework on parenting styles. We did not find a dog-directed parenting style of being permissive or uninvolved, which we attribute to a study population of devoted dog owners and our findings should be interpreted with this specific study population in mind. We found evidence of dog-directed parenting styles and provide a fundament for determining their possible impact on the different aspects of a dog’s life. Strategies of parents to raise their children are recognized as parenting styles, reflecting relatively stable patterns in parenting behaviour and goals the caretaker has with parenting the child. Parenting styles are relevant because of their effects on the development and well-being of children. They could exist also in the owner-dog relationship and, possibly, present a pathway to improve dog behaviour and welfare. The dimensions underlying four main parenting styles , 2, 3 arTo determine the effects of the parenting styles on children, in human psychology the Parenting Styles and Dimensions Questionnaire (PSDQ) is used. The PSDQ was specifically developed to identify parenting practices based on self-reports by parents of (pre)school-aged children . The oriParenting styles influence child development, with the authoritative style being optimal. Academic competence and self-reliance for instance, as measured on a three-point Likert-scale in 4,081 fourteen- to eighteen-year-old US children, was significantly higher in authoritatively parented children than in children parented otherwise . MiscondResearch on the identification of parenting styles and the effects of parenting is abundant in human psychology, but almost non-existent in companion animal sciences . ConsequSufficient argumentation exists to assume a correspondence in behaviour patterns that parents show towards children and dogs, but there is need for further scientific evidence. Here we determined styles in the parenting of dogs by using an (adapted) PSDQ, as the existence of dog-directed parenting styles could bring new ways to improve the owner-dog relationship and latter’s quality of life. We validate the psychological construct of parenting styles in the owner-dog relationship and determine if parenting styles express similarly or dissimilarly in the relationships of owner to dog as in parent to child.Dog owning parents of at least one child (age not reported) participated in a Dutch language web-based survey, after having been recruited via pet stores, vets, dog schools, human schools,  foodbank organisations, online and hardcopy magazines on (human) parenting as well as on (companion) animals. The online survey’s introduction explained the purpose of the research and the study did not involve treatments or interventions in the life of respondents or their dogs. The questionnaire was not repeated, meaning it did not interfere significantly with normal daily life, and did not include questions that were psychologically burdening. This exempts the study from review by our ethics committee, according to the guidelines of Wageningen University Medical Ethics Review Committee . Informed consent was not obtained as respondents choice to participate freely via internet and the purpose of the research was stated at the start of the online survey.The survey ran from May 2016 till November 2016 and included three main parts, with the first consisting of 21 questions (items) on the background of the owner, her/his household and the dog. We did not collect privacy sensitive information for the purpose of our research, such as names and addresses of respondents. The second part contained 62 items on dog-directed parenting, explained below. The third held 62 items on child-directed parenting, being the Parenting Styles and Dimensions Questionnaire (62-PSDQ) developed by Robinson et al.  for measWe wanted to validate the child-directed PSDQ for measuring dog-directed parenting and adapted existing items to situations dog owners encounter when raising their dog. For example, the original item ‘I spank when my child is disobedient’, was reformulated to ‘I use a corrective slap when my dog misbehaves’  to detect possible obscurities in the questions. PSDQ items were measured on a five-point Likert scale, rating the likelihood of scenarios occurring as never (score 0), nearly never (1), neutral , nearly always (3) and always (4). Parenting style scores were calculated following Robinson et al.  by summis) rank, resulting in similar outcomes and only the latter are presented. Descriptive results are presented as means ± standard deviations (s.d.), with median values and lower/upper quartile indicated as additional information on parenting style distribution.Statistical analyses were performed with GenStat (18th edition). Item scores were analysed for associations by standard Principal Component Analyses  based oWe constructed a short Dog-Directed Parenting Styles and Dimensions Questionnaire (DD-PSDQ) based on the statistical outcomes of the first PCA on the dog-directed 32-PSDQ. We dropped four items from this first PCA, retained the other twenty items with loadings ≥ │0.4│. Dropped items associated with a different parenting style than in Robinson’s original PSDQ. On the kept items we ran a second PCA and added two items from the 62-PSDQ to create a more balanced set of scales. The two chosen items had the highest loadings of the items additional to the 32-PSDQ. Internal consistency of the newly constructed DD-PSDQ dimensions was tested with Cronbach’s alpha.N = 470; male: 8%, N = 43) and more than three quarters  had completed upper secondary education or higher. Age of the respondents was indicated in seven age categories and most belonged to age groups 35–44 years  and 45–54 years . The mean (±s.d.) number of children was 1.7±0.8 on a four-point scale of one to four or more. Most respondents had one child  or two , 9% (N = 45) had three children and 4% (N = 19) had four or more.Dog owning parents of at least one child (age not specified) participated in the study. The number of respondents was 518, but with the occasional items remaining blank . In the following, percentages are given relative to this total and the precise sample sizes are indicated. The majority of the respondents was female , 28% (N = 144) had two, 9% (N = 44) had three and 5% (N = 27) had four or more dogs. Dogs were of varying breeds and the vast majority of dogs had been purchased in part for companionship  and/or walking , 24% (N = 126) had been bought, also, for partaking in dog sports, 13% (N = 67) for the owner to feel safer, 7% (N = 38) for breeding, 7% (N = 37) for work, 6% (N = 30) for dog shows, 4% (N = 23) for animal assisted therapy and 5% (N = 25) for guarding and/or as resident (‘yard-kept’) dog, meaning the dog resides mainly at the premises, not indoors. Nearly three quarters of the respondents’ dogs were reported to be always inside the house when the owner was inside , for 23% (N = 121) this was ‘mostly’ and for 4% (N = 16) this was ‘nearly never’ or ‘never’.The mean (±s.d.) number of dogs held by respondents was 1.6±0.9, again on a four-point scale of one to four or more. More than half of the respondents had one dog  and dog-directed parenting .Credibility of the 32-item Parenting Styles and Dimensions Questionnaire (PSDQ) for measuring the three basic parenting styles was confirmed by the analyses of scores for both child- and dog-directed parenting styles. The measures of 32-PSDQ and 62-PSDQ correlated significantly for both child-directed parenting . For the three basic styles, the correspondence between parenting children and dogs explained in the range of 19–35% of the variation and for the uninvolved style 10–24% of the variation was explained.Dog-directed parenting and child-directed parenting styles correlated significantly (32-PSDQ: authoritarian rN = 518) parented their children and dogs  with sets of 32 and 62 items on the everyday ways in which dog owning parents  we run a second PCA on the dog-directed 32-PSDQ items shown in s = 0.94  for 32-PSDQ authoritarian and DD-PSDQ correction orientated, rs = 0.88 (P<0.001) for 32-PSDQ authoritative and DD-PSDQ intrinsic value orientated, and rs = 0.65 (P<0.001) for 32-PSDQ authoritative and DD-PSDQ training orientated. These relatively high correlations indicate that measures by the DD-PSDQ connect to the original measuring tool (with consistencies as well as marked differences) and, likely, the underlying theoretical framework.Next, to create a more balanced number of items across three main dimensions, we added the two authoritative-training orientated items from the PCA on the 62-PSDQ that had the highest loadings of the items additional to the 32-PSDQ. These being ‘I practice certain behaviour with my dog before asking this behaviour in a more difficult situation’ (loading: 0.70) and ‘I channel my dog's misbehaviour into a more acceptable activity’ (0.60). So, within the assumed dog-directed style of authoritarian parenting, the use of voice and physical contact varied independently, but for reasons of compatibility with the existing theoretical framework we decided to merge the two independent components into one authoritarian-correction oriented style. To us, these two styles do not reflect differences in the dimension of demandingness, but merely in the way of expressing it verbally or physically. Thus, our end DD-PSDQ consisted of 20 items, eight measuring the authoritarian-correction orientated style, six the authoritative-intrinsic value orientated style and six the authoritative-training orientated style . Tests fHere we show how the concept of child-directed parenting styles applies to dog-directed parenting, with distinct differences like the separation into two authoritative styles. We found an expected style of authoritarian dog-directed parenting, but likely our study population of devoted dog owners prevented us from detecting styles of permissive or uninvolved parenting. Adapting an existing child-directed PSDQ for use with dogs without adding any new items facilitates that expected parenting styles resurface in the data, which makes our finding of differences in parenting dimensions directed towards children and dogs especially salient.We confirm that the 32-item Parenting Styles and Dimensions Questionnaire (32-PSDQ) is a valid alternative to its lengthier counterpart of 62 items and demonstrate how it can be re-constructed into a 20-item Dog-Directed Parenting Styles and Dimensions Questionnaire (DD-PSDQ) that shows good internal consistency on its three scales and associates logically with the original 32-PSDQ dimensions of authoritarian and authoritative parenting.Our finding of dog-directed parenting styles, at least for authoritarian and authoritative parenting, expands on earlier research indicating that dog owners typically experience strong and family-like bonds with their dogs , 17. SimThe two dog-directed parenting styles characterized by low demandingness, i.e. permissiveness and uninvolvedness, remained undetected in our analysis. It is important to realise that this does not exclude their existence. We suspect our study population to have held only few dog owners with a permissive or uninvolved parenting style as completing the lengthy survey, which was necessary to address the different aspects for developing a DD-PSDQ, took some effort and commitment. The permissive and uninvolved style are known for making relatively little effort in parenting  and peopIn our measurement of the parenting styles, we deployed the 32-PSDQ and 62-PSDQ and confirmed the shorter 32-PSDQ as a valid tool amongst the several parenting style measurements that have been used in (human directed) research . The 62-We constructed a 20-item DD-PSDQ that is compact, requires little effort to complete and captures variation in an authoritarian-correction orientated style and in two authoritative styles, with a connection proven to the original 32-PSDQ measuring tool and theoretical framework. For capturing the full spectrum of four parenting styles the 32-PSDQ may yet prove to be the better tool though. As explained, the DD-PSDQ does not capture distinct styles of parenting permissively or uninvolved. Further research with a broad spectrum of dog owners, including those who are less inclined to partake in studies, is needed to determine the existence of permissive and uninvolved styles in dog-directed parenting. Until this is resolved, we suggest to base research on dog-directed parenting on both the 32-PSDQ and the DD-PSDQ. The 32-PSDQ has the potential to capture all four parenting styles, should the styles of permissiveness and uninvolvedness prove to be relevant in dog-directed parenting. The DD-PSDQ is useful especially in measuring variation in dog-directed parenting among common dog owners who vary in responsiveness and to at least some degree in demandingness, though extremes of low demandingness may be missed.Being able to assess dog-directed parenting styles is a first step to promote appropriate parenting in dogs. In humans, the authoritative parenting style is known to give optimal child outcomes, in terms of high school/academic performance , 12 and S1 AppendixPSDQ items in the uninvolved style following Blakely Kimble (2009) and Baumrind (2013)(PDF)Click here for additional data file.S1 TableResults of loadings after rotation from PCA-analysis for 32/62-PSDQ  and new dog-directed parenting styles in 518 Dutch dog owning parents; presented are the loadings ≥ |0.4|.(PDF)Click here for additional data file.S2 TableDutch dog owning parents (N = 518) reported on child-directed parenting in 32 items adapted from the Parenting Styles and Dimensions Questionnaire (PSDQ). Answers on a five-point Likert scale were analysed by Principal Component Analysis and presented are the loadings ≥ |0.4| and percentages of variation explained by the main components, which represented dimensions of parenting authoritatively, authoritarian and permissively.(PDF)Click here for additional data file.S3 TableDutch dog owning parents (N = 518) reported on dog-directed parenting in 62 items adapted from the Parenting Styles and Dimensions Questionnaire (PSDQ). Answers on a five-point Likert scale were analysed by Principal Component Analysis and presented are the loadings ≥ |0.4| and percentages of variation explained by the main components, which represented dimensions of parenting authoritatively and authoritarian.(PDF)Click here for additional data file.S4 TableDutch dog owners (N = 518) filled out a 32-item Parenting Styles and Dimensions Questionnaire (PSDQ) adapted for assessing dog-directed parenting styles. Answers on a five-point Likert scale were analysed by Principal Components Analysis, omitting items with loadings < │0.4│for the main components. Presented are the final outcomes on eighteen items with loadings ≥ |0.4| and percentages of variation explained by two components of parenting authoritarian-correction orientated (two times four items), one component of authoritative-intrinsic value orientated (six items) and one of authoritative-training orientated (four items), together explaining 48% of variation.(PDF)Click here for additional data file.


An artemisinin-based combination therapy, artesunate (AS) plus sulphadoxine-pyrimethamine (SP), was compared to SP monotherapy to provide evidence of further treatment options in southern Mozambique.Plasmodium falciparum malaria were randomly allocated SP (25/1.25 mg per kg day 0) or AS/SP . Allocation was concealed, but treatment was open-label except to microscopists. The primary objective was the relative risk of treatment failure, which was assessed using World Health Organization response definitions modified to a 42-day follow-up.Between 2003 and 2005, 411 patients over one year and 10 kg with uncomplicated dhfr/dhps mutation increased the relative hazard of failure compared to fewer mutations  and baseline axillary temperature increased the relative hazard of failure by 50% for each °C increase .Of the 411 subjects enrolled, 359 (87.3%) completed the follow up period . A survival analysis including 408 subjects showed that the polymerase chain reaction-adjusted cure rates were 90.4%  and 98.0% (95% CI 94.8%–99.3%) for SP and AS/SP respectively. Multivariable analysis showed that treatment with AS/SP decreased the relative hazard of treatment failure by 80% compared to SP  and age over seven years decreased the relative hazard of failure by 70% , when compared to younger age. However, having a quintuple While both treatments were efficacious, AS plus SP significantly decreased the relative hazard of treatment failure compared to SP monotherapy Artesunate plus sulphadoxine-pyrimethamine, but not sulphadoxine-pyrimethamine monotherapy, met the current WHO criteria of >95% efficacy for policy implementation.NCT00203736 and NCT00203814 Malaria is responsible for a large health burden, including an estimated 19% of deaths among children under five years of age [Plasmodium falciparum malaria varies throughout the country according to the season, other environmental factors, and the use of vector control measures. A treatment policy with effective anti-malarials is a key component of malaria control programmes and data regarding resistance of parasites to anti-malarial drugs are now a critical factor in drug policy decision-making. While chloroquine was the national policy in Mozambique for the treatment of uncomplicated malaria as recently as 2003, evidence of its poor efficacy throughout Africa since the 1980s signalled a need for alternative anti-malarials [falciparum malaria provided the partner drug is efficacious. ACT deliverers a more rapid cure compared to non-ACT, reducing gametocyte carriage , and delaying the development of anti-malarial resistance [Mozambique  evaluation in Namaacha and Matatuine Districts, Maputo Province, during 2002 and data showed that a combined polymerase chain reaction (PCR)-adjusted cure rate exceeded the 80% level considered for it to be combined with other anti-malarials to sustain its useful therapeutic life [dhfr) and dihydropteroate synthetase (dhps), mutation across all study sites was 5–6% throughout the study period [[There was clear evidence that ACTs substantially reduced treatment failure, recrudescence and gametocyte carriage but this had not been reported for Mozambique . Howevertic life ,9. The p period [, Raman JThis multi-centre, open-label, parallel-group RCT was conducted in Maputo Province, Southern Mozambique in four public-sector health facilities in two phases: initially Catuane and Namaacha (2003), thereafter Boane and Magude (2004–2005) Figure . There hP. falciparum infection by rapid diagnostic test . Patients diagnosed with P. falciparum parasitaemia up to 500,000 asexual parasites/μml blood (on thick Giemsa-stained smear) and with axillary temperature greater than or equal to 37.5°C (or history of fever within the previous 24 hours) were suitable for inclusion. Patients receiving folate or anti-malarials  within seven days were excluded. Also excluded were patients with danger signs  or severely ill, those with a history of G6PD deficiency or allergy to the study (or related) drugs, or those with serious underlying disease [Male and non-pregnant, non-breastfeeding female patients with malaria symptoms, that were older than one year of age and weighed more than 10 kg, were invited to give informed consent prior to screening for  disease ,13.®. Roche, Gauteng, South Africa) or AS/SP  (Table Eligible subjects were randomized to treatment with SP monotherapy (Fansidara) Table . Subjectin vivo therapeutic efficacy of anti-malarials, with extension to a 42 day follow-up due to the long elimination half-life of SP [®), clinical signs and symptoms to capture adverse events, and concomitant medications [The visit schedule was based on the WHO protocol for assessment of fe of SP ,15. Afteications . A fingeP. falciparum DNA extracted from these dried samples based on variations in three highly variable proteins  was used to determine if treatment failure was due to a re-infection or recrudescence of the original infection [dhfr  and dihydropteroate synthetase, dhps  genes were detected using nested PCR and restriction endonuclease cleavage [Asexual parasite density was calculated using the number of asexual parasites and assuming 8,000 leukocytes/μl blood. Genotyping of nfection ,18. Infecleavage . DigestiThe primary study objective was to compare the risk of treatment failure between the treatment groups, adjusted for baseline characteristics. A secondary outcome compared the rate of parasite clearance between groups.Response to treatment was classified according to WHO definitions for low to moderate malaria transmission intensity areas, modified for a 42-day follow up ,15. SubjKey fields were verified by a study monitor and double-entered into an MS Access 2000  database. Laboratory data were recorded electronically. Data were imported into Stata/IC 10.0  when outcomes, time-to-event indicators and explanatory variables were programmed.Major protocol violations were defined as subjects: i) missing days 1, 2, 3 of the ACT arm or days 2,3 of the monotherapy arm; ii) taking folic acid or a concomitant medication with anti-malarial activity ; iii), whose day 42 visit was more than three days late; and iv) taking an incorrect dose or repeat dose for vomiting. Data were not censored for missed visits as it was assumed intensive AE monitoring would detect malaria symptoms or treatment. Missing data fields were dropped where appropriate and subjects lost to follow-up after day 0 were automatically dropped from the time-to-event analyses. An attempt was made to follow all subjects to day 42 despite protocol violations.Sample sizes were calculated assuming an adequate clinical and parasitological response (ACPR) of 75% for SP and 90% for AS/SP  indicating 100 per treatment group in each phase of the study .Time-to-event outcomes, which were considered the most appropriate for the data, were analysed using Kaplan Meier (KM) survival methods and Cox's Proportional Hazards Regression. This allowed data up to the point of loss to follow-up or withdrawal to be included and for the relative hazard to approximate the relative risk.KM distributions and survival curves for the two treatment groups were compared using a log rank test. Should model diagnostics suggest hazards were not proportional, parametric models were applied . Time toThe study was approved by the Ethics Committees of the Mozambican Ministry of Health and the University of Cape Town prior to commencement, and conducted in accordance with the South African Clinical Trials Guidelines 2000 . Staff mSubject disposition is shown in Figure dhfr/dhps mutation increased the relative hazard of failure 3.2 fold compared to fewer mutations. The proportional hazard assumption was satisfied overall; however there was an accelerated time to failure (approximately 6-fold) for subjects with a quintuple mutation compared to subjects with fewer mutations suggesting that early treatment failure is particularly associated with presence of a quintuple mutation. When the analysis was repeated after excluding or censoring for major protocol violations , results confirmed those found above.From the survival analysis, Figure The ACT group had faster parasite clearance rates compared to the SP monotherapy group (log-rank p = 0.001) Figure . Multiva3-105 parasites per asexual life cycle, compared to 10-103 for SP) [The aim of this study was to provide evidence to Mozambican health policy-makers regarding efficacy of SP with or without AS as treatment for uncomplicated malaria. In a survival analysis, both drug regimens were effective, however adding artesunate improved outcomes significantly with regard to clinical and parasitological cure, and peripheral parasite clearance time. These results are consistent with the literature, that suggests combining AS with SP improves efficacy, provided efficacy of SP is sufficiently high . In addiSubjects aged over seven years had a 70% lower risk of treatment failure than those seven and under, and were 30% more likely to clear parasites faster (the latter association was only found when protocol violators were removed). These findings are consistent with the knowledge that immunity to malaria infection increases with age . Howeverdhps) and dihydrofolate reductase (dhfr) enzymes respectively. Resistance to SP develops due to accumulation of point mutations in genes that encode these enzymes. The presence of 5 mutations, 3 in the dhfr gene (S108N/N511/C59R) and 2 in the dhps gene (A437G/K540E) are associated with treatment failure [st line treatment of uncomplicated malaria (AQ-SP replaced with AS-SP) and as intermittent preventive treatment for pregnant women (SP in 2007).Sulphadoxine and pyrimethamine are anti-folates that inhibit the dihydropteroate synthetase  protocol violators, results were very similar. Including data from non-adherent subjects or those taking prohibited concomitant medications is useful for assessing the effect of these common practices on treatment response. AS courses shorter than three days are not recommended due to their lower efficacy and the increased likelihood of anti-malarial drug resistance emerging if combined with the more slowly eliminated SP . SP may,dhfr 164 mutation, associated with high-level pyrimethamine resistance found in neighbouring Malawi [This study found that, while both SP monotherapy and in combination with AS were effective, the ACT was far superior, supporting its current use in Mozambique as the national policy for the treatment of uncomplicated malaria. However, it is recognized that combinations with SP may have a limited useful life due to the spread of parasite resistance, and inadequate drug levels young children achieve. It is also possible that SP efficacy has declined since this study due to the national implementation of SP as an intermittent preventive treatment policy for pregnant women during 2007. Of further concern is the presence of the g Malawi .The authors declare that they have no competing interests.EA participated in study design, coordinated the study, conducted the statistical analysis and drafted the manuscript, TC managed the Mozambique study teams, YC provided medical expertise to the study teams, FL participated in study design, calculated sample sizes and supervised the statistical analysis, JR conducted molecular analyses, AB supervised analysis and reporting. KIB, Principal Investigator of the South East African Combination Anti-malarial Treatment (SEACAT) Evaluation, conceived of, and designed the study, and contributed to the analysis. All authors read and approved the final manuscript.


In 2004, a major outbreak of hepatitis A among tourists returning from Egypt involved 351 case-patients from 9 European countries who were infected with a single strain (genotype 1b). The case-control study identified orange juice as the most likely infection vehicle. Vaccination against hepatitis A virus is strongly recommended before travel to disease-endemic areas. In Germany, approximately half of the 1,400–2,300 cases of laboratory-confirmed HA reported annually since 2001 were acquired abroad. In mid-August 2004, infectious disease surveillance in Germany showed a strong increase of HAV infections in tourists returning from Egypt, where HA is highly endemic (The line listing of HA patients included persons with laboratory evidence of recent HAV infection (anti-HAV immunoglobulin M [IgM]) who stayed at hotel X after June 1, 2004. Also listed were hotel guests with HA disease , without laboratory confirmation, who had traveled with persons with laboratory-confirmed cases.A case-control study was performed among hotel X guests >17 years of age residing in 3 German states. The time span between the earliest arriving case-patient's last day at hotel X and the latest arriving case-patient's first day there was defined as the "minimum period of transmission" (MPT). Case-patients came from the line listing. Healthy hotel guests who stayed at the hotel during the MPT who had neither been vaccinated against HAV nor previously infected with HAV were eligible as controls. Telephone interviews were conducted with a standardized questionnaire that elicited information on demographic factors, foods and drink consumption, and participation in recreational activities such as swimming, day trips, etc.2 tests) and multiple logistic regression were performed ; p<0.05 was considered statistically significant. The Egyptian authorities' investigation included testing all hotel employees for HAV antibodies , and scrutiny of food suppliers. Serum samples were obtained from German case-patients for testing by reverse transcription–PCR (RT-PCR) in the VP1–2A junction and sequencing of a 160-bp-long PCR fragment of the VP1 region.For statistical analysis, exposures were dichotomized into "ever" versus "never," and the "number of days exposed" was calculated. Univariate analysis  persons. Case-patients were 2 to 67 years of age (median 34 years) and 54% were male. Overall, 47% of case-patients were hospitalized. Risk of hospitalization rose with increasing age (p for trend = 0.001). The MPT lasted from June 24 to July 23 . No moreSixty-nine HA case-patients (60% response among the 115 case-patients in the 3 states) and 36 controls were included in the statistical analysis. Eighty-seven percent of the case-patients reported absence from work for 3 to 56 days (median 26 days), and 54% of the case-patients were hospitalized for 2 to 25 days (median 9 days).Case-patients and controls did not differ significantly by age, sex, recreational activities, consumption of ice cream or salads, or other foods consumed or behavioral characteristics. Case-patients were significantly more likely to have drunk orange juice served at the breakfast buffet (82%) than were controls (64%) . In multivariate analysis, no other exposures were retained. A dose-response relationship became apparent between number of days of orange juice consumption and HA . Case-paThe on-site investigations in Egypt did not identify hotel staff positive for HAV IgM. Minimal fluctuation among hotel staff renders it unlikely that an HAV-positive employee was missed. Investigations at the orange juice producing plant found significant hygiene problems. In addition, the finished product did not undergo heat treatment. This producer did not cater to other hotel chains in Hurghada.This large outbreak demonstrates risk and clinical impact of HA for nonimmune travelers to HA-endemic countries. In Germany, the outbreak accounted for 12% of all HA case-patients notified in the year 2004.The results of the outbreak investigation strongly point to orange juice as the infection vehicle. In the case-control study, among a broad range of foodstuff, beverages, and recreational activities queried, the consumption of orange juice was the only exposure significantly associated with HA, with higher doses of juice significantly increasing HA risk. These findings are corroborated by the inspection of the hygienic conditions under which the juice was produced in Egypt. The juice was most likely contaminated during the manufacturing process, e.g., by an infected worker with imperfect hand hygiene or by contact of fruit or machinery with sewage-contaminated water.,–>15 days (Citrus fruit and citrus juices have only rarely been implicated as vehicles of HA outbreaks, with contamination typically described during preparation just before consumption (The fact that juice was consumed by 60% of healthy controls may be explained in part by fluctuating virus concentration within the juice, which resulted in varying degrees of infectiousness during the 4-week period. A contaminated lot may have been phased-in and out slowly by gradual mixture with other lots. Also, the study design did not allow the exclusion of controls who did not know they were immune.The Hurghada outbreak-strain clearly differs from strains that have caused nontravel-associated outbreaks in Germany in recent years. Two large autochthonous outbreaks were caused by HAV type 1a strains (Vaccination against HA is recommended for all nonimmune travelers to HA-endemic areas (


Isoproterenol-induced cardiac hypertrophy in mice has been used in a number of studies to model human cardiac disease. In this study, we compared the transcriptional response of the heart in this model to other animal models of heart failure, as well as to the transcriptional response of human hearts suffering heart failure.We performed microarray analyses on RNA from mice with isoproterenol-induced cardiac hypertrophy and mice with exercise-induced physiological hypertrophy and identified 865 and 2,534 genes that were significantly altered in pathological and physiological cardiac hypertrophy models, respectively. We compared our results to 18 different microarray data sets  representing various other animal models and four human cardiac diseases and identified a canonical set of 64 genes that are generally altered in failing hearts. We also produced a pairwise similarity matrix to illustrate relatedness of animal models with human heart disease and identified ischemia as the human condition that most resembles isoproterenol treatment.The overall patterns of gene expression are consistent with observed structural and molecular differences between normal and maladaptive cardiac hypertrophy and support a role for the immune system (or immune cell infiltration) in the pathology of stress-induced hypertrophy. Cross-study comparisons such as the results presented here provide targets for further research of cardiac disease that might generally apply to maladaptive cardiac stresses and are also a means of identifying which animal models best recapitulate human disease at the transcriptional level. Physiological increase in cardiac muscle mass occurs normally during development, during pregnancy, and in response to sustained exercise . ConversFor example, some of the animal models of heart failure are induced by pharmacologic agents to induce a direct cardiomyopathy; however, it is relatively rare for the human heart to be injured through such a mechanism . Even thGenetic expression array analysis allows the characterization of tissue by assessing the expression patterns of thousands of genes. Such investigations of diseased human myocardium have been used to elucidate which molecular pathways are altered in particular etiologies of heart failure ,10. In sWe induced cardiac hypertrophy in mice using isoproterenol, which has been shown to induce significant cardiac dysfunction associated with a high mortality rate (up to 80%) . We subjIsoproteronol (ISO) administration and chronic exercise training successfully created cardiac hypertrophy in experimental animals. As shown in Fig. To determine whether the increase in heart size was due to increased myocyte size or increased numbers of cardiomyocytes, we quantified cell cross-sectional area in H&E stained heart cryosections. Each cell examined had a circularity (height/width) = 0.90, and > 400 cells were measured for each sample type . As expected, myocyte cross-sectional area was significantly increased in mice treated with ISO, compared to sedentary control mice Fig. , and carTo assess and compare the global transcriptional profiles of mice with pathological and physiological cardiac hypertrophy, we performed gene expression microarray analysis of RNA from the left ventricles of ISO-treated mice, mice that were exercise trained, and sedentary control animals. The experiments were performed in triplicate, and a separate microarray ran for each individual mouse heart. A gene was considered as differentially expressed if the fold-change was at least 1.5, the Benjamini and Hochberg-corrected (B-H) p value was less than 0.05, and the alteration occurred for each of the 9 possible pairwise comparisons . Based on these criteria, there was a total of 940 probe sets representing 865 different genes (634 up-regulated and 231 down-regulated) that were significantly and reproducibly altered between control and ISO-treated animals. The transcriptional profile of mice that were swam twice daily, on the other hand, was more profoundly affected, with 2,670 probe sets representing 2,534 genes altered , compared to sedentary control animals. Eight genes were selected for verification by real-time reverse transcriptase polymerase chain reaction (RT-PCR), the results of which validated the microarray analysis (Table -20 to 6.6 × 10-4) and cellular growth and proliferation for mice that were swam . The top physiological functions in ISO-treated mice were immune system development and function  and immune response , whereas the most over-represented physiological functions in exercised mice were organismal survival  and organ development . Molecular pathway analysis also indicated that cardiovascular system development and function  was a major physiological function associated with genes altered in exercised mice, but not in ISO-treated animals. The top canonical signalling pathways in ISO-treated mice included acute phase response , fibrosis , NRF2-mediated oxidative stress response , and VEGF signalling . The most over-represented signalling pathways identified by Ingenuity for the list of genes altered in response to exercise were PI3K/AKT , ERK/MAPK , integrin , PPAR signalling , and PPARα/RXRα activation .Based on pathway analysis results using Ingenuity Pathway Analysis software, the top associated signalling network functions in mice treated with ISO were cardiovascular disease, free radical scavenging, and molecular transport (28 genes out of 39 network molecules), whereas the top associated network functions for mice that exercised were gene expression, cell morphology, and cell-cell signalling and interaction (35 genes out of 40 network molecules). Interestingly, the most over-represented molecular functions after multiple hypothesis testing correction were cell death for ISO-treated mice  and hematological system development and function . Of these 159 overlapping genes, 139 were altered in the same direction in pathological and physiological cardiac hypertrophy models . Conversely, there were 20 genes that were altered in each condition in the opposite direction   However, there were 159 genes that were altered by both types of cardiac hypertrophy, whose functions were mainly related to immune response, particularly antigen presentation , myocar403/+ , aortic 403/+ -24 in va403/+ , as well403/+ . We also403/+ . For Aff403/+ ,17, raw 403/+ mice, rats with aortic banding, drug-induced cardiac hypertrophy, myocardial infarction, arteriovenous shunt, and high salt diet), and 4 human diseases .As expected, there was a great deal of variation in the total numbers of altered genes between the various studies, array platforms, disease models, strains, and time frames. It is perhaps worth noting, however, that our intention was not to highlight potential differences in experimental technique between groups or compare the potential value of one disease model over another. Rather, we hoped to identify a set of genes that would represent those canonical changes that are captured most frequently under various experimental conditions and thus might represent consistent genetic expression alterations that might be applicable to the study of the human end-stage failing heart. We therefore grouped study types together and considered the most profound alteration (i.e. largest fold-change in magnitude) as representative for that group. Groups were chosen based on model type and animal, while more detailed parameters  were not considered. This resulted in 14 distinct experimental groups with relevant cardiac expression data: 2 exercise models (swimming mice and rats trained on treadmills), 8 pathological models  that were altered in our study and in both mice and rats irrespective of the model type or study. Col3a1 was of particular interest, because it was down-regulated in response to exercise in mice  and rats (1.8-fold) but up-regulated in most pathological cardiac hypertrophy models, including αMHC403/+ mice (2.2-fold), aortic binding in mice (3.1-fold) and rats (1.6-fold), arteriovenous shunt (1.7-fold), myocardial infarction (2.5-fold), and idiopathic and ischemic cardiac hypertrophy in humans . Interestingly, Col3a1 was down-regulated (2.9-fold) in Dahl rats fed a high salt diet, but only at the latest time point tested (15 wk). There were two exercise-induced genes that differed in directionality depending upon which type of rodent or experimental model was used. Timp3 was up-regulated in swimming mice but down-regulated in exercised rats. Conversely, dbp was up-regulated in rats and down-regulated in mice  altered in exercised mice and rats. Moreover, the PI3K/AKT was statistically over-represented in the 57 genes consistently altered across experimental models, based on Ingenuity pathway analysis . Over-represented cellular processes included cell death , cellular assembly and organization , molecular transport , and cellular development . Immune response  was the most significantly over-represented physiological function.Consistent with molecular signalling events known to occur under conditions of physiological cardiac hypertrophy, there were three PI3K/AKT signalling pathway molecules (-7 to 6.3 × 10-3), cell-cell signalling and interaction , cell death , cellular movement , and cellular development . Twelve of the genes were specifically associated with cardiovascular system development and function .We used a similar approach to examine ISO-induced transcriptional alterations, expecting that each gene was similarly altered for at least one human disease equivalent to be considered significant. The results of this comparison, including fold-changes obtained from other animal models and also exercised rodents, are shown in Table To identify genes that are most likely to be relevant to human disease, we compared the microarray results from our study along with all the previous studies listed in Table -7 to 5.1 × 10-3), cellular movement , cellular growth and proliferation , cell-to-cell signalling and interaction , and cell cycle . The most over-represented physiological functions were tissue morphology , cardiovascular system development and function , connective tissue development and function , skeletal and muscular system development and function , and organismal development . The most profoundly affected signalling network, involving 23 out of the 64 genes broadly associated with animal cardiac hypertrophy models and the human disease indicated that TGF-β in particular might be generally involved in the dysfunctional heart . When the four lists were compared, there were 64 genes that were significantly altered in at least two of the human diseases that also exhibited significant overlap (based on the aforementioned criteria) with the animal models . The two data sets for which raw data were unavailable were excluded. Similarity was measured as the number of genes in common with fold-changes that occurred in the same direction  with a magnitude of at least 1.5. All possible pairwise comparisons were performed, and the resulting matrix was used to create a clustering diagram to illustrate which groups most closely resembled one another Fig. . As expeA variety of methods have been used to simulate human heart disease in animals, and a great many studies have been conducted to examine the transcriptional profiles of these animals, especially in conjunction with signalling pathway perturbation or drug discovery. Few studies, however, have attempted to coalesce these various data to identify canonical molecular signalling pathways. A notable exception is Strom et al , who comThe results of this study show that ISO induces transcriptional alterations in mouse hearts that differ drastically from those elicited by normal hypertrophy in response to exercise training . All experimental procedures for this study were approved by the Institutional Animal Care and Use Committee at UTSW. Pathological cardiac hypertrophy was investigated using the isoproterenol-induced subacute myocardial injury model as previously described [-1.d-1, dissolved in 0.9% NaCl was inserted into the infrascapular subcutaneous tissue and the incision sutured. After 10 days of ISO administration, mice were sacrificed, and left ventricles were collected and processed for subsequent assays.Eight-week old male C57BL/6 mice were purchased from the Jackson Laboratory and housed in the Animal facility at University of Texas Southwestern Medical Center (UTSW), Dallas, TX, in accordance with the standards set forth in the escribed . BrieflyExperimental physiological cardiac hypertrophy was induced via exercise, as previously described . BrieflyHearts were excised from mice following euthanasia, grossly trimmed in frontal orientation, blotted free of excess blood and embedded in Tissue Freezing Medium . Rapid freezing of embeds was achieved by partial immersion in liquid-nitrogen-supercooled isopentane prior to storage at -80°C. At a later time, embeds were equilibrated to -24°C and sectioned on a Leica CM3000 cryostat . Eight-micron slices were adhered to silanated slide glass  and air-dried. Resulting sections were stained with routine hematoxylin and eosin (H&E) for histopathological analysis or with Masson trichrome to assess fibrosis. In order to assess apoptosis, TUNEL (TdT-mediated dUTP nick end labeling) assays were performed using the DeadEnd Fluorometric TUNEL System, as described by the manufacturer . Specimens were photomicrographed in bright field on a Leica DM2000 microscope , equipped with an Optronics Microfire CCD camera . Image acquisition was conducted using Optronics PictureFrame 2.0 software  and morphometric analysis was conducted using Image J 1.38w software  for Macintosh computers.http://www.ncbi.nlm.nih.gov/geo database (Accession GSE18801).Microarray sample preparation and analysis was performed as previously described. Briefly, total RNA from left ventricles of experimental animals was isolated using TRIzol Reagent  per manufacturer's instructions and purified by phenol-chloroform extraction and ethanol precipitation. RNA (20 μg) was further processed and hybridized to the GeneChip Mouse Genome 430 2.0 Array  by the Microarray Core Facility at University of Texas Southwestern Medical Center per manufacturer's instructions. Data were analyzed using GeneSifter  and Spotfire DecisionSite 9.0 . Briefly, data were normalized using robust-multi average (RMA) method, and signals for each group were averaged before performing Student's t test with Benjamini and Hochberg correction and pairwise comparisons for sedentary mice versus mice that received ISO treatment or were exercised. Genes were considered as altered if the folds-change was at least 1.5 and adjusted p value ≤ 0.05. Consistency was also expected, which was assessed by performing all possible pairwise comparisons of individual samples. A fold-change of at least 1.5 was expected for each replicate comparison , and this alteration was expected to be at least 50% greater than the fold-change derived from comparison of any two replicate samples . This effectively eliminated differences that might arise as a result of natural biological variation between mice. Raw and processed data  were deposited in the Gene Expression Omnibus (GEO) online http://www.ncbi.nlm.nih.gov/geo/. Gene expression data from autopsied human hearts and failing hearts collected during heart transplant surgeries were also obtained from Cardiogenomics PGA [For comparison of our gene expression results to previous studies, raw microarray data (CEL or text files) from mice or rats with pathological or physiological cardiac hypertrophy induced by a variety of methods  to results obtained from the various other animal and human studies, a perl script was written to match genes across the various lists by Accession number (for same species results) or gene symbol (for inter-species results comparisons). If neither the Accession number nor gene symbol matched perfectly, then the gene descriptions were compared and same identify was considered if the names differed by nor more than 25%. If the ISO or swim results contained more than one match for a single data point in a published study, only the top three matches were recorded, with the best match ordered first. This perl script was later modified to compute averages for the multiple matches. This method would be expected to fail to indentify some homologous genes in which there are name designation differences between species . However, our intention was to greatly minimize false positive and thus compare only those genes that were truly the same between the various data sets. To ensure that this was indeed the case, all final genes lists representing overlap between study results were also manually examined and any genes that were computationally misidentified subsequently removed.To produce a similarity matrix, average fold change was calculated for all animal model and human disease samples, compared to its appropriate control after normalization (e.g. ischemic heart compared to non-diseased autopsied heart). The two studies for which raw data were unavailable were excluded . For comparison for the two cardiac hypertrophy models used in this study (ISO-treated and swimming mice compared to sedentary controls), normalized signal values were used for clustering after Z score transformation. The similarity matrix was clustered using normalized pairwise scores as described above using CLUSFAVOR 6.0 .Hierarchical clustering of genes was performed using Cluster/Treeview -polymerase chain reaction (PCR) was performed in the iCycler iQ  using SYBR Green I dye , as previously described . Brieflyt test or ANOVA. Differences were considered statistically significant for p < 0.05.Values presented are expressed as mean ± S.E.M. All comparisons between groups were performed using a one-tailed Student's CLG oversaw the project, carried out the microarray analysis and subsequent interpretation of data, participated in the mouse, histology, and real-time RT-PCR experiments, and drafted the manuscript. MAS aided in experimental design, data interpretation, and manuscript preparation and also provided medical expertise. ME carried out the initial animal experiments and participated in subsequent sample collection. LDO participated in microarray results interpretation, experimental design, and sample processing. DAW performed animal surgeries, exercised mice, took animal readings, and collected animal samples. JL performed histology, real-time RT-PCR, and cell area counts. JFM assisted with histology, data interpretation, and manuscript preparation. LJM wrote and utilized computer scripts for analysis of previously published array data. TQP participated in animal experiments and took animal readings. NMK participated in the interpretation of experimental results and in editing of the manuscript. HRG participated in the design and coordination of the study and helped to draft the manuscript. All authors read and approved the final manuscript.Supplementary Table 1 . The file provided is an Excel format that includes gene IDs, gene names and symbols, biological functions, fold-changes, statistical values.Click here for file


To evaluate real-world pharmacologic treatment of mixed dyslipidemia in patients with diabetes mellitus (DM).All commercial health plan members in a large US managed care database with complete lipid panel results  between 1/1/2006 and 12/31/2006 were identified . DM patients  with mixed dyslipidemia were defined as having any 2 suboptimal lipid parameters . Lipid treatment status 6 months pre- and post-index date was determined using pharmacy claims for any lipid therapy.Post-index, 41.1% of DM patients with 2 abnormal lipid parameters and 45.1% with 3 abnormal lipid parameters did not receive lipid-modifying treatment. Post-index treatment rates were 57.4%, 63.6%, and 66.4% for patients with LDL-C, HDL-C, and TG in the most severe quartiles, respectively. Statin monotherapy was the primary lipid-modifying regimen prescribed . Less than 30% of treated patients received combination therapy.Over 40% of DM patients with mixed dyslipidemia received no lipid-modifying therapy during the follow-up period. Those who were treated were primarily prescribed statin monotherapy. This study suggests that DM patients are not being treated to ADA-suggested targets. Cardiovascular disease (CVD) is a primary cause of morbidity and mortality among patients with hyperglycemia or type 2 diabetes mellitus (DM) despite the availability of effective therapies to treat major risk factors such as elevated blood pressure and cholesterol levels ,2. The mThe burgeoning prevalence of insulin resistance throughout the world is greatly increasing the incidence of mixed dyslipidemia. It is estimated that approximately 40% of patients with coronary artery disease have low-density lipoprotein cholesterol (LDL-C) levels below 130 mg/dL yet these patients also have low levels of HDL-C, with or without increased levels of triglycerides (TG) ,10. OverObservational studies indicate that low HDL-C levels are strongly and independently associated with increased CHD risk -13. The Elevated serum levels of TG are an independent risk factor for CHD even after adjustment for HDL-C . In a suMultiple guidelines writing groups advocate combination therapy for the management of multiple lipid abnormalities  ,19. StatDespite the abundance of data confirming the role of suboptimal lipoprotein levels as a risk factor for CHD and the availability of guidelines enumerating therapeutic lifestyle modifications and pharmaceutical treatment options for the management of mixed dyslipidemia, a substantial proportion of dyslipidemic patients remain untreated or inappropriately treated ,25, or pThis was a retrospective claims data analysis using medical and pharmacy data, laboratory results, and enrollment information from a large managed health care plan in the United States, with the largest concentration of patients being in the southern and midwestern regions. Claims for services provided to members of this health plan are submitted for payment by physicians, facilities, and pharmacies. At the time the study was conducted, the administrative claims database included data for approximately 14 million health plan enrollees with both medical and pharmacy benefits. All study data were de-identified and accessed with protocols compliant with the Health Insurance Portability and Accountability Act. Institutional Review Board approval was therefore not required for this study.This study was conducted to determine lipid treatment patterns in DM patients with mixed dyslipidemia. Study patients included commercial health plan enrollees with a laboratory value for LDL-C, HDL-C, and TG all drawn on the same day during the time period from January 1, 2006 through December 31, 2006. An index date was set as of the date of the first suboptimal test result or first optimal test result. Patients were required to have been continuously enrolled for 182 days prior to and 182 days following the index date.Two groups of patients were created: a subset with all lipid values  under control  and another subset with at least 1 suboptimal lipid value . The criteria for the suboptimal cohort were developed in accordance with the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) III  and AmerPatient demographic variables  were captured from the enrollment data. Lipid lab values  were obtained from the lab results data. Study patients were observed for 182 days prior to the index date (pre-index period) and for 182 days after the index date (post-index period) to determine their lipid risk factors for CVD and their pharmaceutical prescription patterns. For the assignment of LDL-C goal, patients were classified into 4 risk categories: (1) age/gender ; (2) CHD ; (3) hypertension ; and (4) DM .®]) and without a claim for other lipid-modifying medications; (2) pharmacy claim for a fibrate  and without a claim for other lipid-modifying medications; (3) pharmacy claim for a niacin/nicotinic acid and without a claim for other lipid-modifying medications; (4) pharmacy claim for "other" medication  and without a claim for other lipid-modifying medications; (5) pharmacy claim for a statin and a fibrate; (6) pharmacy claim for a statin and niacin/nicotinic acid; (7) pharmacy claim for a statin and other lipid-modifying medication; (8) pharmacy claim for a fibrate and a niacin; (9) pharmacy claim for a fibrate and other medications ; (10) pharmacy claim for a niacin and other medications ; (11) combinations other than those already mentioned; and (12) no claim for a lipid-modifying medication.The presence of a pharmacy claim for any lipid therapy, including statins, fibrates, niacin, or various combinations, was determined. Specifically, separate determinations were made to identify patients with the following prescription patterns during the pre-index and post-index periods: (1) pharmacy claim for a statin  met the definition of having DM, and 82.3%  of the DM patients had at least 1 suboptimal lipid value and 17.7%  had optimal values for all lipid tests The mean age of the study population was 52 years, and was similar in the suboptimal cohort and optimal cohort (p = 0.0725). Fifty-two percent of patients were female and 47% were male in the study population , approximately 54%  had more than 1 lipid abnormality. The lipid abnormalities in the DM population were as follows: 23% had high LDL-C values only, 13% had low HDL-C only, 10% had elevated TG only, 10% had high LDL-C and low HDL-C, 12% had elevated LDL-C and TG, 16% had low HDL-C and elevated TG, and 16% had abnormal values for all 3 lipid fractions.Overall, an increase in lipid-modifying therapy from the pre-index period to the post-index period was observed in DM patients with mixed dyslipidemia. In the pre-index period, 68% of DM patients with suboptimal LDL-C and HDL-C, 58% with suboptimal LDL-C and TG, 32% with suboptimal HDL-C and TG, and 60% with suboptimal values for all 3 lipid parameters received no treatment for dyslipidemia. In the post-index period, the percentages of patients not receiving therapy decreased to 58%, 43%, 29%, and 45% in each group, respectively. Patients with suboptimal HDL-C and TG had the smallest decrease in the percentage of patients not receiving lipid-modifying therapy Figure .Among DM patients with more than 1 lipid abnormality, 25% to 37% were prescribed statin monotherapy, 3–6% received fibrate monotherapy, and less than 1% received niacin monotherapy during the post-index period. Figure The treatment patterns for patients with DM were also evaluated by quartiles of suboptimal LDL-C, HDL-C, and TG values. The ranges for the quartiles are defined in Figure Despite the availability of treatment guidelines for dyslipidemia management and the abundance of clinical trial evidence highlighting the benefits of lipid-modifying therapy to reduce the risk for CHD and its clinical sequelae, treatment of DM patients with abnormal LDL-C, HDL-C, and TG levels continues to be suboptimal. In our patient population, 16% of patients with suboptimal lipid values and 21% of our mixed dyslipidemia group had DM. Our DM patient population had a high (> 50%) proportion of patients with abnormal HDL-C and TG levels. This is in line with the results of a recent study by Grant et al. , who alsA large percentage of DM patients with suboptimal lipid levels in our study did not receive lipid-modifying therapy either prior to or after a suboptimal lipid value was obtained . While the percentage of patients receiving lipid-modifying therapy increased in the post-index period, a substantial number of DM patients with suboptimal lipid levels did not receive any lipid-modifying therapy in the post-index period irrespective of abnormal lipid parameter, or the degree of the abnormal value. At least 30% of DM patients with suboptimal lipid levels across all mixed dyslipidemic groups were not treated with any lipid-modifying medication for at least 6 months post-index. Although lipid treatment rates increased post-index in all mixed dyslipidemic groups, and even though treatment rates were correlated with the number of total risk factors, abnormal lipid parameters and more severe lipid value quartiles, the incremental use of lipid-modifying medication was not as substantial as would be expected, given the compelling association between multiple lipid abnormalities and cardiovascular risk in patients with DM. Of particular note is that in DM patients with low HDL-C and elevated TG, the most common post-index lipid therapy used was statin monotherapy (in 37% of patients), and only 17% received any niacin or fibrate therapy to target suboptimal HDL-C or TG. Grant et al.  describeThese results are also consistent with those reported by Klingman and colleagues , who evaAlthough the reasons for the suboptimal treatment of dyslipidemia are beyond the scope of this study, several explanations have been proposed to account for the discrepancy between guideline recommendations and real-world treatment patterns. These include suboptimal patient-follow-up, use of low-potency statins in high-risk patients, inaccurate pill counts and refill records, patients' inability to pay for medications, lack of patient motivation , physiciNCEP ATPIII recommends adjunctive therapy with niacin or fibrates  for dyslipidemic patients with multiple CVD risk factors who have low HDL-C and/or high TG, after the LDL-C goal has been achieved with statin monotherapy ,19,32. OOur findings regarding prescribing habits for mixed dyslipidemia are consistent with results reported by Stacy and colleagues , who evaThe findings of this study must be considered within the limitations of the data and study design. Claims data are collected for the purpose of payment and not research and may not accurately capture a patient's medical use history. While these data provide insight into real-world treatment patterns, they are subject to possible coding errors. Certain information is not readily available in claims data that could have an effect on study outcomes, such as certain clinical and disease-specific parameters.Furthermore, the presence of a prescription claim does not necessarily indicate that the drug was taken or that it was taken as prescribed – nor does it reflect those patients who may have received drugs without the presence of a prescription claim either by receiving drug samples or filling a prescription outside of the health care pharmacy system. The presence of a diagnosis code on a medical claim does not demonstrate positive presence of disease, as it may be incorrectly coded or included as a rule-out criterion rather than as an indication of actual disease.In addition, because patients were identified from a managed care plan, this study may not be generalizable to the general population and may not be applicable to a setting outside of managed care. Finally, a limitation specific to this study is that the description for values in the laboratory results does not clearly indicate whether lipid values were obtained during fasting. Although these limitations do not reduce the strength of the study, they must be considered when interpreting the results.In real-world clinical practice, pharmacologic treatment rates increased only slightly upon assessment of multiple lipid abnormalities and in patients with DM. Over 42% of DM patients with mixed dyslipidemia received no lipid-modifying therapy after suboptimal lipid levels were ascertained, and those patients who were treated, even those with low HDL-C and high TG levels who may benefit from adjunctive treatment with niacin or fibrates, were primarily prescribed statin monotherapy. These study results suggest DM patients are not being treated to ADA-suggested targets. Further research is warranted to investigate how physicians tailor lipid-modifying therapy in these patients so that solutions aimed at improving treatment rates can be devised in order to help DM patients better achieve guideline-defined LDL-C, HDL-C, and TG goals, thereby potentially reducing their risk for cardiovascular events.DL works for Abbott Laboratories, which provided the financial support to sponsor the research for this study. PT received an honorarium from Abbott Laboratories for consulting services on this study and development of this manuscript.DL conceived of the study and participated in its design and coordination. PT participated in the design of the study and helped draft the manuscript. JS participated in the design of the study and performed the statistical analysis and programming. VZ participated in the design and coordination of the study and drafted the manuscript. All authors read and approved the final manuscript.


The most frequently used molecular imaging technique is currently 18F-deoxy-glucose (FDG) positron emission tomography (PET). FDG-PET holds promise in the evaluation of recurrent or residual ovarian cancer when CA125 levels are rising and conventional imaging, such as ultrasound, CT, or MRI, is inconclusive or negative. Recently, integrated PET/CT, in which a full-ring-detector clinical PET scanner and a multidetector helical CT scanner are combined, has enabled the acquisition of both metabolic and anatomic imaging data using one device in a single diagnostic session. This can also provide precise anatomic localization of suspicious areas of increased FDG uptake and rule out false-positive PET findings. FDG-PET/CT is an accurate modality for assessing primary and recurrent ovarian cancer and may affect management. FDG-PET/CT may provide benefits for detection of recurrent of ovarian cancer and improve surgical planning. And FDG-PET has been shown to predict response to neoadjuvant chemotherapy and survival in advanced ovarian cancer. This review focuses on the role of FDG-PET and FDG-PET/CT in the management of patients with ovarian cancer. Recently, we have evaluated 16α-18F-fluoro-17β-estradiol (FES)-PET, which detects estrogen receptors. In a preliminary study we reported that FES-PET provides information useful for assessing ER status in advanced ovarian cancer. This new information may expand treatment choice for such patients. Ovarian cancer is the second most common gynecologic malignancy. It has a relatively poor prognosis, accounting for approximately half of all deaths related to gynecologic cancer . Conventin vivo. The most commonly used radioisotope tracer is 18F-deoxy-glucose (FDG), a glucose analog which is preferentially taken up by and retained within malignant cells. Depending on the area or organ under study, baseline glucose metabolism may be low, further highlighting the difference between normal background tissue and tumor fluoro-17β-estradiol (FES) is a radiopharmaceutical that binds to the estrogen receptor (ER), thereby demonstrating the existence of this receptor [16α-Ffluoro-1receptor , as a fuFDG is excreted through the urinary tract and also physiologically accumulates in the bowel. Intense activity in the urinary or gastrointestinal tract can interfere with the optimal evaluation of the abdomen and pelvis. The most simple solution to this is to request that the patient empties their bladder just prior to imaging and to initiate imaging from the pelvis, before the bladder is full .Other useful techniques to avoid false positives are bladder catheterization and furosemide administration. Koyama et al. reported that continuous bladder irrigation is useful for eliminating FDG activity in the bladder during FDG-PET (FDG activity in the urinary tract was eliminated in 80% of patients). The technique had satisfactory diagnostic utility with 100% sensitivity, 86% specificity and 98% accuracy for differentiating malignant from nonmalignant lesions. However, there is no foolproof method for avoiding bowel uptake .It is now hoped that FDG-PET/CT will increase both sensitivity and specificity of PET by identifying physiologic tracer uptake and delineating cancerous lesions with low or absent FDG uptake a round or oval area and was noted as an intense focal abnormality singular with a SUV greater than 3.0. It would therefore seem difficult to distinguish focal FDG uptake in the normal ovary from that in malignant lesions [Increased physiologic ovarian FDG uptake in menstruating patients has been reported as an incidental finding. Lerman et al. evaluated patterns of FDG uptake during 4 phases of the menstrual cycle in 246 pre- and postmenopausal women without gynecologic tumors. Increased ovarian uptake was detected in 21 premenopausal patients, of whom 15 were at mid cycle and 3 reported oligomenorrhea. An ovarian standardized uptake value (SUV) of 7.9 differentiated benign from malignant uptake with a sensitivity of 57% and specificity of 95% . Nishiza lesions . MoreoveConventional morphological imaging modalities including US, CT, and MRI have been widely used to determine whether a suspicious ovarian tumor is malignant -3. US peColor and pulse Doppler techniques may aid in the diagnosis of ovarian cancer. Buy et al. compared gray-scale ultrasound with duplex and color Doppler in the evaluation of adnexal masses. Adding color Doppler to gray-scale morphologic information increased specificity from 82% to 97% and increased positive predictive value (PPV) from 63% to 97%, but duplex Doppler indices provided no further information -27. A laCT and MRI have been utilized to further evaluate ovarian masses -3. AlthoThe sensitivity of FDG-PET in the detection of ovarian cancer was 78% in our study ; this waIn screening for ovarian cancer, US is the most important modality. MRI or FDG-PET, in addition to US, can provide further information about ovarian tumors and improve specificity. However, our study showed that the addition of FDG-PET to MRI does not yield additional information for the diagnosis of ovarian masses after US .Recently, Castellucci et al. assessed the accuracy of FDG-PET/CT in distinguishing malignant from benign pelvic lesions, compared with transvaginal ultrasonography (TVUS). Adding FDG-PET/CT increased specificity from 61% to 100%, negative predictive value (NPV) from 78% to 81%, PPV from 80% to 100%, and accuracy from 80% to 92%. They concluded that FDG-PET/CT provides additional information to TVUS in the differential diagnosis of benign from malignant pelvic lesions . In concA major problem in ovarian cancer is that a high proportion (75%) of patients have advanced stage disease at the time of diagnosis, which results in a 5-year survival rate of only 41% . ThereafOur study is the first to show that the addition of FDG-PET to CT improves the staging accuracy of ovarian cancer . The reaIn conclusion, FDG-PET is a useful and promising tool but not an established procedure in the staging of ovarian cancer patients. As studies in this field have been small-scale and have had variable results, a multicenter study with more data and showing clinical utility for routine use is needed before the procedure can be applied routinely for patients with confirmed or suspected ovarian cancer ,39-43.Recurrent ovarian cancer is almost never curable, but early detection of recurrence theoretically increases the chance that salvage treatment will result in prolonged remission and sustained quality of life. Conventional imaging modalities often give nonspecific results and are suboptimal for the reliable detection of peritoneal recurrence. The identification of more accurate imaging modalities should improve management decisions for patients with recurrent ovarian cancer.In 2002 we reported that FDG-PET was useful for following up an ovarian cancer patient in whom the only feature suspicious of recurrence was a rising CA125 level within the normal range . HavrileThe use of FDG-PET/CT for detecting recurrent ovarian cancer was first described by Makhija et al. in 2002 . In 2008In conclusion, FDG-PET may provide benefits for those with elevated CA-125 (>35 U/ml), CT- or MRI-defined localized recurrence amenable to local destructive procedures, and clinically suspected recurrent or persistent cancer when biopsy cannot be performed. Using FDG-PET/CT or PET/contrast-enhanced CT is reported to have higher sensitivity and specificity than FDG-PET alone for detecting recurrent disease. We have summarized sensitivity and specificity for each imaging modality for the diagnosis of primary and recurrent/metastatic ovarian cancer in Tables SUV is the most common PET parameter measured in the clinical setting. Its calculation is simple, and most contemporary FDG-PET/CT scanners display the imaging in these units, provided the injected dose and the patient weight have been entered when setting up the PET acquisition . The rolWe have previously assessed whether FDG-PET is useful for assessing malignant activity and gathering prognostic information in ovarian cancer . We evalA study of GLUT-1 expression in ovarian carcinoma by Canturia et al. showed that GLUT-1 status is an independent prognostic factor of response to chemotherapy in advanced ovarian carcinoma, and that patients over-expressing this marker have a significantly shorter disease-free survival rate . FurtherIn conclusion, glucose consumption, as determined by analysis of SUV in FDG-PET, may be a non-invasive biomarker that can predict response to chemotherapy and survival in ovarian cancer.Patients with advanced ovarian cancer who have completed a planned course of chemotherapy have frequently undergone a systematic surgical exploration and may be asymptomatic. About 36% to 73% of patients may have persistent disease detected at second-look procedures. Patients with residual disease should undergo continuous adjunctive therapy, while those without disease may discontinue adjunctive therapy. The cost-effectiveness and value of FDG-PET as a substitute for a second-look procedure have therefore been explored ,60,61. AThe sensitivity of ovarian cancer to hormonal therapy has a real, although modest, role in the treatment of advanced ovarian cancers resistant to chemotherapy. Many agents have been evaluated, including antiestrogens, estrogens, progesterones, androgens, aromatase inhibitors, and gonadotropin releasing hormone agonists (GnRH). As anticancer agents, hormonal therapies produce an approximate 10% response rate in previously treated patients. A correlation may exist between the presence of hormone receptors and a response to therapy .Here, we first showed that FES uptake was observed at primary and metastatic sites in three cases of advanced ovarian cancer. In these patients, we compared FES uptake and immunohistochemistry results for surgical specimens from patients with both primary and metastatic sites. These data indicated that FES uptake in PET was associated with ER status, particularly ER-α status, in ovarian cancer. A representative case was that of a 66-year-old woman with huge uterine leiomyoma and ovarian serous adenocarcinoma who underwent FES-PET before and after cytoreduction surgery. Before surgery, FES-PET showed moderately increased uptake in both the leiomyoma and ovarian cancer regions; the maximum SUV was 2.5 and 2.1 figure . Hence FFDG-PET holds promise in the evaluation of cancer spread or recurrent or residual disease when other radiographic data are uncertain. FDG-PET/CT might be a useful supplemental investigation to detect primary and recurrent ovarian cancer earlier than FDG-PET and other conventional imaging tools. In addition, FES-PET may have the potential to provide useful information about hormone status in advanced ovarian cancer.The authors declare that they have no competing interests.YY drafted the manuscript. TK, TT, OH, and FK conceptualized, edited, and revised the manuscript. All authors have read and approved the final manuscript.


Assessment of coronary heart disease (CHD) risk is typically based on a weighted combination of standard risk factors. We sought to determine the extent to which a lipidomic approach based on red blood cell fatty acid (RBC-FA) profiles could discriminate acute coronary syndrome (ACS) cases from controls, and to compare RBC-FA discrimination with that based on standard risk factors.rds derivation set and validated on the remaining 1/3rd. The area under receiver operating characteristic (ROC) curves (c-statistics), misclassification rates, and model calibrations were used to evaluate the individual and combined models. The FA discriminated cases from controls better than the SRF  and the FA profile added significantly to the standard model . Hosmer-Lemeshow calibration was poor for the FA model alone (p = 0.01), but acceptable for both the SRF (p = 0.30) and combined models (p = 0.22). Misclassification rates were 23%, 29% and 20% for FA, the SRF, and the combined models, respectively.RBC-FA profiles were measured in 668 ACS cases and 680 age-, race- and gender-matched controls. Multivariable logistic regression models based on FA profiles (FA) and standard risk factors (SRF) were developed on a random 2/3RBC-FA profiles contribute significantly to the discrimination of ACS cases, especially when combined with standard risk factors. The utility of FA patterns in risk prediction warrants further investigation. Several recent risk prediction algorithms have been proposed, such as those from the Prospective Cardiovascular Munster (PROCAM) study e.g., plasma triglycerides and non-esterified FAs) are sensitive indicators of acute changes in dietary habits and in hepatic and adipocyte function. Adipose tissue FA composition is a long-term (months to years) reflection of dietary habits, whereas membrane FA composition  provides a more intermediate estimate (weeks). We Fatty acids (FAs) are powerful modulators of cell membrane receptors and affect signal transduction, gene transcription, and eicosanoid metabolism. They are present in many tissue compartments, including plasma , adipose tissue and cell membranes. Some of these compartments  excluded symptomatic ischemic heart disease or confirmed an alternative explanation for their presentation . Three physicians reviewed the charts of all patients for whom diagnostic uncertainty remained and attained consensus on the final diagnosis. With this approach, a total of 1,661 patients were included in this registry and enrolled as described in All consecutive patients admitted to two hospitals associated with the University of Missouri-Kansas City School of Medicine were prospectively screened for an ACS between March 2001and June 2004 . The subPatients having blood drawn for routine clinical testing were recruited from blood drawing centers at Saint Luke's Hospital (where 88% of the cases were derived) between March 2004 to March 2005 as outlined in ACS patients completed a baseline interview within 24 to 72 hours of admission, and detailed information on patient presentation, race, comorbidities, and treatments were obtained by chart abstraction. Standard risk factors included age, sex, total-C, HDL-C, a history of diagnosed hypertension and diabetes, and smoking status RBC-FA composition was measured as previously described 768 patients diagnosed with ACS were matched one-to-one with controls on the basis of age (5-yr windows), gender, and race (Caucasian vs. non-Caucasian). 228 were excluded due to incomplete information on HDL-C, total-C, self-reported hypertension (HTN), self-reported diabetes mellitus (DM), age, gender, or current smoking status . Two-thiStepwise unconditional multivariable logistic regression was used to develop prediction models with p = 0.01 used to enter and remain in the model. One model was developed using RBC-FAs(FA), another with the 7 standard risk factors (SRF), and another using the FAs selected in the FA model combined with the standard risk factors (SRF+FA). Natural log transformations were used for HDL-C and total-C to improve normality. Robust, non-parametric 95% confidence intervals (CI) of the parameter estimates were obtained using bootstrapping method with 10,000 replicates from the training data set for both FA models. In addition to using the stepwise selected FAs, four pre-specified FA metrics were also tested for their ability to add to the SRF model: the omega-3 index (eicosapentaenoic acid (EPA)+docosahexaenoic acid (DHA)) iβ, i = 0 to p) multiplied by each subject's FA levels  or by the standard risk factors  as follows:For each MLR model a single continuous variable, a risk score, was calculated (equation 1) as the linear combination of the parameter estimates  was determined for each model and the difference compared to the SRF alone. The standard error (SE) for the c-statistic was computed as described by Hanley and McNeil Due to matching on age, sex and race, there were no differences in these attributes . As expeOdds ratios for the 7 standard risk factors alone, FAs alone and the combination are presented in Using the standard risk factors, and the parameter estimates for blood cell FAs, the ability of MLR models to discriminate cases from controls were compared, both alone and in combination . The FA i.e., p>0.05) were those that included the SFR, either alone or when combined with FAs  trans-oleic (or elaidic) acid and eicosadienoic acid. Associations of increased intakes and/or in vivo levels of industrially-produced trans FAs with CHD risk are well established The relations between risk for CHD and RBC levels of the individual FAs included in the model generally fit well with previous observations: inverse associations with omega-3 and omega-6 FAs and direct associations with trans FAs. Of the ten FAs included in the model, increasing levels of eight were inversely associated with odds for ACS case status. These included the FAs of both the omega-3 and omega-6 series, the monounsaturated FA palmitoleic acid, and the saturated FA, stearic acid. These levels reflect a mixture of diet and metabolism since the essential FAs (omega-3 and omega-6) are strongly affected by diet whereas palmitic and stearic, which can be synthesized de novo, reflect metabolic processes. Direct associations were found only with A potential weakness of case control studies is that the exposure of interest could be altered by the clinical event it is intended to predict. The use of RBC-FAs is attractive in this regard as levels remain stable for at least 4–6 weeks and are not appreciably affected by CHD events in primate models e.g. discrimination, positive likelihood ratios, misclassification rates, etc.), FA profiles performed well and show promise as a new risk marker for CHD. Other proposed criteria such as the potential to reveal novel disease mechanisms are also satisfied since FAs affect a variety of metabolic and regulatory pathways linked to CHD . These may be in part mediated by alterations in the activity of membrane-associated receptorse.g., inflammatory) Based on those criteria set forth by Vasan Strengths of this study include a large sample size, a rigorously-defined ACS population, detailed FA analysis, the use of stable biomarker of tissue FA status, use of separate derivation and validation data sets, and a comprehensive examination of several metrics of model utility. Potential limitations should also be considered. It is possible that the outpatients who agreed to participate were not truly representative of the case population. Nevertheless, the fact that the standard risk factors predicted case-control status very comparably to other prospective studies suggests that control selection bias was unlikely to have materially affected our results. Hypertension and diabetes were self-reported in controls. Since these conditions are often under-diagnosed some controls may have incorrectly reported normal blood pressure or glycemia. Under-reporting by controls would help the SRF, but not the FA, model discrimination. In addition, FA profiles have been reported to predict the presence of vascular disease independently of hypertension and diabetes In conclusion, an RBC-FA lipidomic approach added substantially to standard risk factors for prediction of ACS. These findings suggest that substantial, previously unrecognized biological information may reside in membrane FA patterns. A deeper appreciation of the mechanisms by which FAs modulate cellular metabolism could lead to a new understanding of causes and pathways of CHD as well as to improved clinical risk prediction and treatment strategies.