Purpose. To evaluate the effect of polydextrose (PDX) on Ca bioavailability and prevention of loss of bone mass. Methods. Twenty-four two-month-old ovariectomized rats were fed three isocaloric diets only varied in fiber source and content up to 60 days . A SHAM group was included as control. Apparent Ca absorption percentage (%ABS), changes in total skeleton bone mineral content (tsBMC) and bone mineral density (BMD) and femur BMD, % Bone Volume, Ca and organic femur content, caecal weight, and pH were evaluated. Results. %ABS and caecum weight of PDX and FOS were higher, and caecum pH was lower compared to OVX and SHAM. PDX reached a higher pH and lower caecum weight than FOS possibly because PDX is not completely fermented in the colon. Changes in tsBMC and femur BMD in FOS and PDX were significant lower than SHAM but significantly higher than OVX. % Bone Volume and femur % of Ca in PDX were significantly higher than OVX and FOS but lower than SHAM. Conclusions. PDX increased Ca absorption and prevented bone loss in OVX rats. Although an optimal calcium intake (CaI) is vital throughout the life cycle, a great percentage of population consumes levels that are far below the recommended amounts. Ca nutrition adequacy is necessary for both bone accretion during growth to achieve an optimum peak bone mass and maintenance in the adult life to suppress bone turnover and, therefore, bone loss , 2. BoneAlthough the increment in CaI would be the most effective strategy for avoiding Ca deficit, if the intake remains inadequate, the ability to improve absorption becomes an important tool to optimize bone health. Ca absorption average only 30% in the adult and, regardless of the CaI, losses through endogenous secretions approximate 120 mg/d .  Nondigestible oligosaccharides can modify the colonic microbiota by increasing the proliferation and activity of beneficial flora  which in Fiber is an essential nutrient in a healthy diet, contributing to health maintenance and preventing the occurrence of different diseases. Polydextrose (PDX), a compound widely used as an additive for more than 15 years, is a soluble fiber, that is, not digested in the upper gastrointestinal tract , 11. The The adult ovariectomized (OVX) rat is a suitable animal model to mimic the estrogen withdrawal of postmenopausal women , 13. Bon On these bases, the aim of the present report was to evaluate the effect of feeding an adequate Ca diet containing 5 g/kg of PDX on Ca absorption and retention into bone in young-OVX rats. In addition, the effect of this diet in preventing the loss of bone mass in OVX rats was compared with the same Ca diet containing a commercial mixture of short- and long-chain fructo-oligosaccharide (FOS) products (Synergy) whose effects on these studied parameters are well documented.n = 8/group) during a 60-day period (SHAM groups: rats were fed a semisynthetic diet prepared according to the American Institute of Nutrition Diet (AIN 93 M) ;OVX group: rats were fed a semisynthetic diet prepared according to the American Institute of Nutrition Diet (AIN 93 M);FOS group (FOS): rats were fed the AIN 93 M diet containing 10 g/100 g diet of Synergy  replacing equal amounts of cornstarch and sucrose;R, Danisco) replacing equal amounts of cornstarch and sucrose.PDX group (PDX): rats were fed the AIN 93 M containing 5 g/100 g diet of polydextrose  were obtained from the Laboratory Animal Service of the School of Biochemistry and Pharmacy, Buenos Aires University (Argentina). Throughout the experiment, animals were allowed free access to deionized water and food and were housed in individual stainless steel cages in a temperature- (21 ± 1°C) and humidity- (60 ± 10%) controlled room with a 12 h light/dark cycle. They were fed a commercial stock diet for laboratory rats during 7 days for acclimatization . After that, ovariectomy was performed by a dorsal approach under light anesthesia (0.1 mg/100 g body weight (BW) of ketamine hydrochloride + 0.1 mg/100 g BW of acepromazine maleate)  and randomly placed on one of the 3 following diets  and phosphorus (P) (0.3%). BW was recorded once a week throughout the study. Food intakes were recorded every three days throughout the experiment, and the total intake, daily intake, and daily CaI were then calculated. This study was carried out in accordance with the National Institute of Health Guide for the Care and Use of Laboratory Animals and approved by the Committee of Health Guide for the Care and Use of Laboratory Animals of the Facultad de Farmacia y Bioquímica, Universidad de Buenos Aires. Food intake and feces recorded during the last three days of the experiment were used to calculate the apparent Ca absorption (%ABS) as follows: [/CaI] × 100. t = 0) and at the end of the experiment (t = 60), total skeleton bone mineral content (tsBMC) and bone mineral density (tsBMD) were determined “in vivo” under light anesthesia with a total body scanner by dual energy X-ray absorptiometry (DXA) provided with a specifically designed software for small animals  as previously described [At the beginning  to perform bone volume % (BV%) on the central area of the metaphyseal bone displayed on the digitalized image [At the end of the experience, rats were placed under anesthesia , and the right femur was excised at sacrifice for biochemical analysis. The right tibia was resected and fixed by immersion in buffered formalin for 48 h, decalcified in 10% ethylene-diamine tetraacetic acid (EDTA) (pH 7) during 25 days, and embedded in paraffin. An 8- to 10-ed image . After the rats were killed, the caecum from each rat was excised, weighed, and split open, and the pH recorded. Feces were dried under infrared light and pounded. Diets and feces were wet ashes with nitric acid using Parr bombs . Femurs were cleaned of any adhering soft tissue and dried at 100°C for 72 hours, and fat was extracted by immersion for 15 days in chloroform-methanol (3 : 1) mixture which was removed and replaced every three days. Finally, it was dried for 48 hours at 100°C. The fat-free and dried femurs were weighed and ashes were obtained at 700°C until white and crystalline. Thereafter, they were dissolved in HCl and diluted for Ca and P analysis. The amounts of Ca and P were calculated as total content and percentage content of dried fat-free tissue, and the femur Ca/P ratio was also calculated.  Ca concentration in diets, feces, and femur was determined using an atomic absorption spectrophotometer . LanthanP < 0.05), intragroup comparisons were tested by Tukey test.Results were expressed as mean ± standard deviation (SD). Differences were tested by one-way analysis of variance (ANOVA) using the INSTAT package. When ANOVA presented statistical differences  throughout the study; BW was also significantly lower in OVX versus FOS and PDX groups from the 1st to the 5th week of the study (P < 0.01) without differences between them. Thereafter, no significant differences among the 3 OVX groups were found until the end of the experiment (8th week). During the studied period, the BW gain was higher in the 3 OVX groups versus SHAM group (P < 0.05); no differences between PDX and FOS groups were observed which showed a tendency to reach lower values as compared to OVX groups (X groups . P < 0.01) without differences between them; in addition, no differences were observed between OVX and SHAM groups while caecum pH was higher and caecum weight was lower in PDX versus FOS group (P < 0.01)  . t = 0 among groups; however, tsBMC at t = 60 was lower in the 3 OVX groups as compared to SHAM group (P < 0.05) without differences between them. The changes in BMC between t = 60 and t = 0 of FOS and PDX groups were significantly lower than SHAM group (P < 0.05) but significantly higher than OVX group, expressed as total content (P < 0.01) or normalized per BW (P < 0.01) without significant differences between them. The tsBMD and its changes were higher in SHAM than in OVX groups (P < 0.05) and in FOS and PDX than in OVX group (P < 0.01) without differences between them. Femur BMD (fBMD) changes were significantly lower in OVX groups as compared to SHAM group (P < 0.05) and significantly higher in FOS and PDX than in OVX group (P < 0.05), without differences between them. Total skeleton BMC (tsBMC) and BMD (tsBMD) and their changes between the end and the beginning of the study are shown in P < 0.05), and only ashes content reached significance as compared to FOS and PDX groups (P < 0.05) which did not present significant differences between them, but they were also lower than SHAM group (P < 0.05). The ashes/organic content ratio was higher in SHAM than in the 3 OVX groups (P < 0.05) which did not present differences among them while no significant differences were observed in femoral Ca/P ratio.P < 0.01); in addition, PDX and FOS presented a significantly higher value than OVX group (P < 0.05), and there was a tendency to be higher in PDX than in FOS group without reaching significance  often obtained from chicory root, and other FOSs has been extensively studied in several experimental models . In thisIn the present paper the source of fiber in the 3 diets was different. While cellulose was the fiber source of the diet fed by the OVX and SHAM groups (AIN 93 M diet), FOS and PDX diets contained a 10% and 5% of different soluble fibers, a mixture of short- and long-chain products (Synergy) and PDX, respectively. It is important to point out that we performed a preliminary test with a diet containing 10% PDX; however, such diet was not well tolerated by the rats. One possible explanation for this effect would be that PDX is not fully fermented in the colon which induced intolerance and diarrhea. Synergy and PDX induce changes in the intestinal microflora which produces SCFA by fermentation that decrease caecal pH . The low SCFA directly or indirectly participated in Ca absorption. In this regard, Ca acetate and propionate pass across cell membranes even more readily than ionized Ca alone . In addiThe amount of Ca absorption and bone retention depends on several experimental conditions such as age, sex, hormonal status, duration of experiment, and diet composition including level of Ca and fructooligosaccharides. Although the absorption measurements could only evidence changes on Ca metabolism for a short time, determinations such as Ca in bone, bone density, and/or histology results showed a long-term impact. In this regard, it has been shown that inulin-type fructans, under certain conditions including OVX, stimulate Ca absorption and enhance bone Ca content , 28, 44,Although further studies are required, the results of the present report demonstrated that PDX has a prebiotic effect. Moreover, this ingredient given jointly with an adequate amount of Ca optimizes its absorption and bone retention in OVX rats improving bone health. This effect may have important implications in preventing osteoporosis.


Supplemental Digital Content is available in the text. Cardiac mass and volumes are often elevated in athletes, but it is not known whether moderate physical activity is also associated with cardiac dilatation and hypertrophy in a healthy adult population.P<0.0001) and elevated LV and right ventricular volumes . Physical activity had a larger effect on cardiac parameters than systolic blood pressure (0.06≤β≤0.21) and a similar effect to age (−0.20≤β≤−0.31). Increasing physical activity was a risk factor for meeting imaging criteria for LV hypertrophy , LV dilatation , and right ventricular dilatation .In total, 1096 adults  without cardiovascular disease or cardiomyopathy-associated genetic variants underwent cardiac magnetic resonance imaging to determine biventricular volumes and function. Physical activity was assessed using a validated activity questionnaire. The relationship between cardiac parameters and activity was assessed using multiple linear regression adjusting for age, sex, race, and systolic blood pressure. Logistic regression was performed to determine the effect of activity on the likelihood of subjects having cardiac dilatation or hypertrophy according to standard cardiac magnetic resonance normal ranges. Increasing physical activity was associated with greater left ventricular (LV) mass (β=0.23; Exercise-related cardiac remodeling is not confined to athletes, and there is a risk of overdiagnosing cardiac dilatation or hypertrophy in a proportion of active, healthy adults. Misclassification of cardiac disease in healthy adults may lead to erroneous risk stratification, inappropriate management, and unnecessary further investigations. In this study, we examined a healthy adult population without cardiovascular risk factors or genetic variants associated with cardiac disease to determine if typical levels of physical activity were associated with cardiac remodeling.Cardiac chamber enlargement and the capacity to increase cardiac output are hallmarks of athletic training.BaggishSee Editorial by See Clinical Perspectivewww.digital-heart.org) between March 2011 and January 2015. We excluded participants at screening who had known cardiovascular disease or were being treated for hypertension, diabetes mellitus, or hypercholesterolemia, or had a first degree relative with cardiomyopathy. All subjects were sequenced for disease-causing cardiomyopathy and channelopathy genes using a comprehensive sequencing assay,8 and 62 individuals with putative pathogenic genetic variants were excluded.9 Female subjects were excluded if they were pregnant or breastfeeding but were eligible if they took oral contraceptives. Standard published safety contraindications to magnetic resonance imaging were applied.10 All subjects provided written informed consent for participation in the study, which was approved by a research ethics committee.One thousand one hundred fifty-eight healthy adult volunteers  were prospectively recruited by newspaper advertisement as part of the UK Digital Heart Project .Physical activity grading was based on the Copenhagen City Heart Study Leisure Time Physical Activity Questionnaire, performed on the same day as the cardiac magnetic resonance imaging. This questionnaire has been shown to discriminate subjects with respect to maximal oxygen uptake and to predict mortality.12 Blood pressure (BP) was measured after 5 minutes rest in accordance with European Society of Hypertension guidelines13 using a calibrated oscillometric device .All measurements were performed by trained research nurses at the study center. Height and weight were measured without shoes while wearing scrubs. Total body fat mass and lean mass were measured using bioelectrical impedance .14 Images were stored on an open-source database .15 Volumetric analysis of the cine images was performed using CMRtools  following a standard protocol  guidelines.16 These reference ranges are the 95% confidence intervals in an independent healthy adult population obtained using identical cardiac software and analysis methods.18 Indexed volumetric data were left ventricular (LV) mass (LVMi), LV and right ventricular (RV) end-diastolic volumes (LVEDVi and RVEDVi), LV and RV end-systolic volumes, LV and RV stroke volumes (LVSVi and RVSVi), and LV and RV ejection fractions. Cardiac index (CI) was derived as (LVSV×heart rate [HR]) divided by body surface area. Interstudy and interobserver reproducibility were assessed in 20 subjects.Cardiac magnetic resonance (CMR) imaging was performed at a single site on a 1.5T Philips Achieva . A standard clinical protocol for assessing biventricular function and volumes was followed according to published international guidelines.www.r-project.org) using RStudio Server . Categorical variables were expressed as percentages. Because of skewed distributions, continuous variables were expressed as median±interquartile range. Inter-rater reliability was assessed using Krippendorff’s alpha coefficient and interstudy agreement using Kendall’s Tau coefficient. Confidence intervals were calculated using 10 000 bootstrap samples.Data were analyzed in R .The Jonckheere–Terpstra test for ordered alternatives was used to assess if there was a statistically significant trend of higher median LVMi, LVEDVi and RVEDVi, indexed LV end-systolic volume and indexed RV end-systolic volume, LV concentricity, LV SVi and RV SVi, CI, and lower median HR and EF, with higher levels of physical activity. The differences between other physical and demographic parameters with respect to activity was assessed with the Kruskal–Wallis H Test. Differences between each individual activity level were then assessed using post hoc pairwise Wilcoxon tests, Bonferroni-corrected for multiple comparisons . The independent variable was physical activity with covariates of age, sex, ethnicity, and systolic BP. Ethnicity was dummy-coded with the largest group, white, as the reference.The associations between activity level and categorical variables (presence/absence of dilatation or hypertrophy) were assessed with Chi-squared tests and logistic regression models, again adjusted for age, sex, race, and systolic BP.P value <0.05 was taken as significant. Standardized β-coefficients are reported in the text, and both standardized and unstandardized β-coefficients are provided in the tables.Regression models satisfied the assumptions of linear regression. In all tests, a Data Supplement.All data sets were analyzed and included in the final analysis. Subject characteristics are described in Table P<0.0001), and there was a reduction in exercise level with increasing age (P=0.01).In an average week, 8% of the cohort reported engaging in no regular exercise at all (level I), 41% light activity (level II), 35% moderate physical activity (level III), and 16% >5 hours of exercise per week (level IV). Men reported higher activity levels than women (Table P<0.0001). There was also a significant pattern of increasing LV concentricity (LV mass to LV EDV ratio) with increasing activity (P=0.003). Resting HR decreased with increasing activity (P<0.0001). Consistent with the increased SV but decreased resting HR, there was no significant association between resting CI and activity level (P=0.94). There was a significant pattern of decreasing LV and RV EF as activity level increased .There was a statistically significant pattern of higher LVMi, biventricular volumes, and stroke volumes  with higher levels of physical activity . For each increase in activity level (I–IV), LVMi increased by 3.7 g/m2, LVEDVi by 4.1 mL/m2, and indexed RVEDVi by 5.0 mL/m2.After adjustment for age, sex, ethnicity, and systolic BP, LVMi and biventricular volumes remained positively associated with activity , RVEDVi , and LVMi  compared with activity level. Age and activity showed a showed a similar strength of association with cardiac parameters .By way of comparison, systolic BP had a weaker association with LVEDVi . Each increase in activity level was associated with an increase in LVSVi of 2.2 mL/m2 and RVSVi of 2.3 mL/m2. HR had a negative association with activity , and each activity level was associated with a 3.6 bpm decrease in resting HR. Overall, there was a weak negative association of activity and CI . LV and RV ejection fractions showed weak negative associations with increasing activity . There was no association between concentricity (LV mass to LV EDV ratio) and physical activity after adjustment for potential confounders .LVSVi and RVSVi were positively associated with activity after adjustment for potential confounders .To ensure that these results were not driven by a small number of true athletes within the >5 hours per week of exercise (level IV) group, we repeated the multiple regression analyses with the exclusion of the highest activity level group and found a similar pattern of remodeling in the level III subjects compared with the other participants. We also compared ratiometric scaling with allometric adjustment for height20 increased with activity level .The proportions of participants classified as having LV hypertrophy, LV dilatation, or RV dilatation by CMR criteriaP<0.0001), LV dilatation , and RV dilatation . There was no significant interaction between sex and activity level . Table In logistic regression models , activity level remained a significant predictor of LV hypertrophy , but the association with LV hypertrophy was no longer significant .As mentioned earlier, to ensure these results were not been driven by a small number of true athletes in the level IV group, we repeated the logistic regression analyses with the exclusion of these subjects. The association of activity with LV and RV dilatation remained significant (adjusted odds ratio 2.4 for LV dilatation [P=0.03), 8.5 for LV dilatation (P=0.001), and 9.1 for RV dilatation (P<0.0001). Similarly, we compared level II subjects with level IV subjects; the resulting adjusted odds ratio was 5.4 for LV hypertrophy (P<0.0001), 5.3 for LV dilatation (P<0.0001), and 6.2 for RV dilatation (P<0.0001).To further understand the association between activity and ventricular geometry, we compared level I subjects with level IV subjects. The resulting adjusted odds ratio was 4.4 for LV hypertrophy .Finally, we repeated the logistic regression analyses with HR, history of current smoking, and history of previous smoking as additional covariates and found the pattern of the results unchanged  surveyed in 2002, our cohort had fewer sedentary adults (8% versus 22%),24 and although participation in athletic sports has substantially increased over the previous decade,5 the rise in overall population activity has been more modest and shows wide geographical variation.31 In contrast to previous studies, participants with genetic variants associated with cardiomyopathy were excluded from this study, ruling out potential genetic confounder effects.9Our cohort of 1096 adults is ≈10× larger than the reference populations used to derive the recommended normal ranges for the CMR community.32 Because this was an observational study, we could only assess the association between variables, but interventional studies have confirmed a causal relationship between both exercise and detraining on cardiac structure and function.33 We performed the most comprehensive sequencing currently available8 and would expect that, if present at all, rare variants outside the genes we sequenced would affect <1 in 25 000 healthy controls.34 We also did not use contrast medium or T1 mapping in this study and so do not know if unsuspected fibrosis was present in those with dilated hearts, although without genetic substrate this would not be expected.35 We did not include RV mass in our analysis because freewall thickness is challenging to measure accurately in healthy volunteers, and it is not a diagnostic criterion for cardiomyopathy36; however, endurance athletes demonstrate a balanced increase in both LV and RV mass, and a similar relationship may be expected in moderate exercise.2 Although we do not have outcome data, the beneficial effects of exercise are clear,37 and regular physical activity may attenuate adverse age-related changes in cardiac structure and function.38 There are reports of extreme exercise being associated with arrhythmias, but it is unlikely that moderate exercise contributes to an arrhythmic substrate in healthy volunteers screened for genetic variants.39Our study has some limitations. The Copenhagen score is a validated and pragmatic approach that clinicians can readily use for assessing activity level. However, more comprehensive surveys, such as those based on the Cross-Cultural Activity Participation Study, provide a detailed breakdown of physical activities, allowing estimation of the metabolic equivalent level.In conclusion, activity-related cardiac remodeling is not confined to athletes and develops at moderate levels of physical exercise typical of a healthy adult population. This physiological adaptation should be considered when assessing adults for heart disease by CMR because the effects of exercise on the heart are of equal or greater importance than those of age or BP.We thank our radiographers Giuliana Durighel, Ben Statton, Marina Quinlan, Catherine Holden, and Alaine Berry, and research nurses Tamara Diamond and Laura Monje Garcia.The study was supported by a Wellcome Trust-GSK Fellowship Grant, by the Medical Research Council, UK, grants from the Fondation Leducq, and by a British Heart Foundation, UK, project grant (PG/12/27/29489). The research was also cofunded by the National Institute for Health Research (NIHR) Biomedical Research Centre based at Imperial College Healthcare NHS Trust, Biomedical Research Unit in Cardiovascular Disease at Royal Brompton & Harefield NHS Foundation Trust and Imperial College London. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, or the Department of Health.None.


Objective. To determine the prevalence and clinical association of anti-phosphatidylserine/prothrombin antibodies (aPS/PT) in patients with a history of pregnancy complications relevant to antiphospholipid syndrome (APS). Materials and Methods. Two hundred and eleven patients with a history of (a) three or more consecutive miscarriages before 10th week of gestation (WG) (n = 64), (b) death of a morphologically normal fetus beyond 10th WG (n = 72), (c) premature birth of a morphologically normal neonate before 34th WG due to eclampsia, preeclamsia and placental insufficiency (n = 33), and (d) less than three unexplained consecutive miscarriages before 10th WG (n = 42). Subjects sera were analyzed for lupus anticoagulant (LA), anti-cardiolipin (aCL), anti-β2-glycoprotein I (anti-β2GPI), and aPS/PT antibodies. Results. 41/169 (24.3%) of patients were positive for at least one measured aPL. The highest prevalence was found for aPS/PT and aCL  followed by LA (7.7%) and anti-β2GPI (7.1%). 11/169 with APS-related obstetric manifestations had only aPS/PT. 17.8% of patients were positive for LA or aCL and/or anti-β2GPI; however when adding the aPS/PT results, an additional 7% of patients could be evaluated for APS. Conclusion. aPS/PT are associated with recurrent early or late abortions and with premature delivery irrespective of other aPL. Several other “noncriteria” aPL are believed to be associated with APS; however the lack of evidence confirming their diagnostic applicability so far prevents their inclusion into classification criteria. In recent years, many studies demonstrated the association of antiprothrombin antibodies with the pathogenesis of APS ). A 2-sided β2GPI was lower (7%)  and low dose aspirin (LDA) which resulted in a successful pregnancy. Six women had no subsequent pregnancies; three of them experienced successful pregnancy prior to APS pregnancy complications. Two women had unsuccessful subsequent single pregnancies despite the anticoagulant treatment. When adjoined, the aPS/PT results revealed an additional 6.5% (11/169) of patients with adverse pregnancy outcome that was positive for aPL.Every patient positive for any of the tested aPL was tested again at least 12 weeks after their first visit and only permanently elevated levels of aPL were considered as a positive result. Prevalence of all aPL tested is shown in wer (7%) . Eleven The frequency of aPL among 42 patients who experienced less than three miscarriages before 10th WG (and did not meet APS classification criteria) was very low and did not show differences from healthy women.Statistical analyses could not find any association of higher levels of aPL being more strongly associated to adverse pregnancy outcome.P < 0.03, age adjusted)  . In factβ2GPI, and aPS/PT) to obstetric complications characteristic for APS presented as OR with 95% confidence interval. As shown in Age-adjusted analyses were performed in order to estimate the relative risk of positive outcome in different aPL tests . Excluding these 12 patients from logistic regression analyses showed that frequencies of aCL and aPS/PT antibodies were still significantly higher as compared to healthy controls .In our group of 169 patients, 12 (6%) had a history of thrombosis and the prevalence of all tested aPL was higher among them as compared to healthy controls  of patients were aPL positive.We found an overall prevalence of aPS/PT of 13.0%, aCL of 12.4%, LA, and anti-k et al.  who suggk et al. . ConsideWe have also determined the frequency of aPL among 42 patients who experienced less than 3 miscarriages before 10th WG and did not meet APS classification criteria. In line with our expectations the prevalence of all aPL in this group was very low (<7%) and was not different from frequencies found in healthy women.β2GPI and provided an overall aPL frequency estimated as 6% for pregnancy morbidity. Rare studies investigated clinical significance of antiprothrombin antibodies in relation to adverse obstetric outcome. Akimoto et al. [The review of the literature revealed 12 studies dated from 1997 to 2013. More than half of the articles were published before 2006, when new revised classification criteria were accepted. Five studies tested only aPT-A antibodies, which are (according to newer data) less significant for APS , 17, 21.o et al. –16. Berto et al.  found coβ2GPI or LA with any of the adverse pregnancy outcomes. This is in line with conclusions of the systematic review [β2GPI and pregnancy morbidity. Also, in the study by Clark et al. [β2GPI are far more associated with thrombotic APS than obstetric APS, while aCL and aPS/PT appear to be associated with both types of APS.Our study did not confirm the association of either anti-c review , implyink et al. , which iβ2GPI to 0.549 for aCL or aPS/PT  to be 0.688 [The diagnostic accuracy of individual aPL for pregnancy losses defined by APS classification criteria, determined by AUC, was low, ranging from 0.499 for anti-r aPS/PT . In our be 0.688 . Certain in vivo experiments [β2GPI, could enable better evaluation of high-risk APS patients and possibly predict further pregnancy losses. Similarly, Ulcova-Gallova et al. [So far, the mechanism by which antiprothrombin antibodies might be involved in morbidity during pregnancy has not been clarified. However, it is very likely that there are more possible pathways involved. It has been suggested that they may lead to pregnancy loss by the promotion of microvascular placental thrombosis, which was supported by histological findings . Placenteriments . An increriments . On the eriments . There ieriments . A revieeriments  concludea et al.  suggesteaPS/PT are associated with adverse pregnancy outcome irrespective of other antiphospholipid antibodies. Therefore, aPS/PT measurement might improve the evaluation of patients with early recurrent pregnancy loss, undiscovered by other aPL tests. Further studies including a larger number of patients with pregnancy complications and/or reproductive failure and apparently healthy donors are needed to determine the independent effects of various antiphospholipid antibodies, as well as aPS/PT, and confirm their potential use in clinical practice.


Eimeria papillata in mice. The data showed that mice infected with E. papillata and treated with PPE revealed a significant decrease in the output of oocysts in their faeces by day 5 p.i. Infection also induced inflammation and injury of the jejunum. This was evidenced (i) as increases in reactive oxygen species, (ii), as increased neutrophils and decreased lymphocytes in blood (ii) as increased mRNA levels of inducible nitric oxide synthase (iNOS), Bcl-2 gene, and of the cytokines interferon gamma (IFN-γ), tumour necrosis factor-α (TNF-α), and interleukin-1β (IL-1β), and (iv) as downregulation of mucin gene MUC2 mRNA. All these infection-induced parameters were significantly altered during PPE treatment. In particular, PPE counteracted the E. papillata-induced loss of the total antioxidant capacity. Our data indicated that PPE treatment significantly attenuated inflammation and injury of the jejunum induced by E. papillata infections.Coccidiosis is the most prevalent disease causing widespread economic loss, especially in poultry farms. Here, we investigated the effects of pomegranate peel extract (PPE) on the outcome of coccidiosis caused by  Eimeria papillata parasitizes mice and provides a convenient model for studying animal coccidiosis through its intracellular development within the mouse jejunum [Eimeriosis is a parasitic disease in which the parasites not only live inside the animal body but can also survive in wet moist conditions outside of the body. Animals that appear healthy shed the parasites in their stools. Unfortunately, in the right conditions, these parasites can quickly multiply within the intestinal tract causing tissue damage and inducing a severe local and systemic inflammatory response , 2, lead jejunum .Medicinal plants have long been found to be useful in the development of new drugs and continue to play an important role in the drug discovery processes . Such pl Eimeria papillata.Ancient Egyptians realized the benefits of pomegranate as a remedy for a variety of ailments  and, in P. granatum peels were obtained from fruits purchased from a local market in Riyadh, Saudi Arabia. The samples were authenticated by Dr. Jacob Thomas . Pomegranate peel extract was prepared according to the method described by Abdel Moneim [l Moneim , with soSwiss Albino mice were inbred under specified pathogen-free conditions at facilities in the Zoology Department, King Saud University. Only male mice were used for the experiments. These were housed in plastic cages and received a standard diet and water ad libitum. The experiments were approved by the state authorities and followed Saudi Arabian law on animal protection. Eimeria papillata were collected from the faeces of infected mice, surface-sterilized with sodium hypochlorite, and washed at least four times with sterile saline solution before oral inoculation as described previously by Dkhil et al. [3 sporulated oocysts of E. papillata suspended in 100 μL sterile saline solution. Subsequently, fresh faecal pellets were collected every 24 h. The pellets collected from each mouse were weighed and the bedding was changed to eliminate reinfection. Oocyst output was measured as previously described by Dkhil et al. [Oocysts ofl et al. . Each moμL pomegranate peel extract (300 mg/kg) daily over 5 days. The dose and the route of injection were selected on the basis of the previous studies by Dkhil et al. [ E. papillata and treated for five days with either sterile saline solution or pomegranate extract, respectively.The mice were divided into four groups with eight animals per group. The first group was gavaged with saline and served as the control, noninfected, group. The second group was inoculated by oral gavage with 100 l et al. , 11. The E. papillata, blood was collected from all groups. Immediately following blood collection, 5 μL of blood was used to prepare blood smears, which were stained with Giemsa stain for differential cell counts.At day 5 p.i. withFor homogenization, part of the jejunum was weighed and homogenized immediately in order to prepare a 50% (w/v) homogenate in an ice-cold medium containing 50 mM Tris-HCl and 300 mM sucrose. The initial homogenate was centrifuged at 500 ×g for 10 min at 4°C. The supernatant (10%) was diluted with the Tris-sucrose buffer.μL NBT (1.0 mg/mL) was added to the jejunal homogenate from each of the groups, followed by an additional period of incubation for 1 h at 37°C. Solutions were then treated with 100 μL KOH (2 M). NBT reduced/g tissue expressed in nmol was determined through spectrophotometry. If radical-scavenging compounds were present in the tissue, less formazan blue would be formed, thus decreasing the absorption at 570 nm.To determine the generation of reactive oxygen species (ROS), we used a modified assay for the intracellular conversion of nitro blue tetrazolium (NBT) to formazan by superoxide anion. Briefly, 200 2O2 is determined colorimetrically by an enzymatic reaction which involves the conversion of 3,5,dichloro-2-hydroxybenzene sulfonate into a coloured product, the intensity of which is inversely proportional to the total antioxidant amount in the original sample [Total antioxidant capacity was assayed by the colorimetric technique using commercial kits . In brief, the method depends on the ability of antioxidants within the test sample to hydrolyse exogenously added hydrogen peroxide. After the reaction time the remaining Hl sample .μm thicknesses. For histological examinations, sections were stained with haematoxylin and eosin. An immunolocalization technique for Bcl-2 was performed on 3-4 μm thickness sections according to [Small pieces of the jejuna were quickly removed and fixed in neutral buffered formalin. Following fixation, specimens were dehydrated, embedded in wax, and then sectioned to 5 rding to . For negβ (IL-1β), tumour necrosis factor alpha (TNF-α), interferon-γ (IFN-γ), inducible nitric oxide synthase (iNOS), and mucin 2 (MUC2). All primer assays used for qRT-PCR were obtained commercially from Qiagen. PCRs were performed as previously described by Dkhil et al. [Pieces of jejunum were aseptically removed, rapidly frozen, and stored in liquid nitrogen until use. Total RNA was isolated using Trizol (Invitrogen). RNA samples were treated with DNase  for at least 1 h and were then converted into cDNA by following the manufacturer's protocol using the reverse transcription kit . Quantitative real-time PCR (qRT-PCR) was performed using the ABI Prism 7500HT sequence detection system  with SYBR green PCR master mix from Qiagen . We investigated the genes encoding the mRNAs for the following proteins: interleukin-1l et al. .Results were expressed as the means ± standard deviation (SD). Data for multiple variable comparisons were analysed by one-way analysis of variance (ANOVA). For the comparison of significance between groups, Duncan's test was used as a post hoc test according to the statistical package program (SPSS version 17.0). E. papillata resulted in an output of about 310,000 E. papillata oocysts per gram of faeces on day 5 p.i., as shown in The presence of infection was associated with clinical signs such as general weakness, poor performance, loss of appetite, and diarrhoea. These signs appear attenuated in the group pretreated with PPE. Infection of mice with E. papillata in the jejunum  induced in vivo production of oxygen free radicals in the jejunal tissues of mice as indicated by NBT assay ( E. papillata infection from 0.3 ± 0.09 to 0.47 ± 0.08 mmol/g (BT assay . When miBT assay . Moreove8 mmol/g . E. papillata (Then, we checked the role of PPE in infection-induced apoptosis, through the histochemical staining of mice jejuna. Immunohistochemical investigation for Bcl-2 showed that PPE was able to decrease the immunoreactivity in the jejuna of mice infected withapillata . Also, Papillata .A significant systemic inflammatory response was initiated as a consequence of the infection, as revealed by the elevation in the number of neutrophils and a reduction in the number of lymphocytes . TreatmeIn this study, the mRNA expression of MUC2 appeared significantly downregulated upon infection but significantly increased again after treatment with PPE .β, TNF-α, and IFN-γ ( E. papillata, whereas PPE significantly reduced the expression of these genes (Also, the mRNA levels of iNOS and inflammatory cytokines, IL-1nd IFN-γ , were upse genes . E. papillata oocysts within the faeces of the infected mice. This diminished output suggests that pomegranate impairs the development of parasites in the host before the relatively inert oocysts are formed and finally released. The fact that pomegranate possesses anticoccidial activity has also been reported by Dkhil [ E. papillata oocyst output in faeces of mice.Pomegranate is one of those candidate plants which have a chemoprotective effect and strong antioxidant potential . The comby Dkhil , where iElevated levels of ROS are an indication of the oxidative stress and cytotoxicity induced by the infection. Our results show, however, that ROS production was decreased significantly by pomegranate administration. A possible explanation is that, at the cellular level, the antioxidant effect of pomegranate may have an impact on the ROS levels induced by the infection by scavenging oxygen free radicals . Increas E. papillata [Natural products have been used for dietary therapy for several millennia and some of these products allegedly exhibit significant antioxidant activity . Also, tapillata . Here, w Eimeria. This may relate to the critical role of neutrophils and lymphocytes during intestinal epithelium invasion by Eimeria [In the present study, the percentage of neutrophils and lymphocytes was subject to significant alterations due to infection with the highly pathogenic Eimeria .Our qRT-PCR results revealed that the expression of MUC-2 was significantly reduced following infection. MUC-2 is the first line of innate host defence in preventing pathogen-induced epithelial injury . PPE wasE. papillata infection-induced status of oxidative damage to the jejuna tissue of mice [ of mice , 10. Nit of mice . This fi of mice . E. papillata are characterized by inflammation, whose treatment with pomegranate appears to reduce. Indeed, pomegranate greatly influences the mRNAs of the inflammatory cytokines IL-1β, TNF-α, and INF-γ mRNA. Of these, IFN-γ is considered to be a major host defence mechanism against primary infections with E. papillata [ E. papillata.The jejuna of mice infected withapillata . It has apillata . In this Eimeria and it is, therefore, highly recommended for use as a food additive in poultry farms.Collectively, pomegranate exhibits a significant anticoccidial, antioxidant, and anti-inflammatory activities serving to protect against the tissue injuries induced by


UGT1A1⁎28 polymorphism associated with Gilbert's syndrome was also found in this Argentine patient. The predominant clinical presentation resembled an acute cholestatic hepatitis associated with severe hemosiderosis, a different and new pattern of PEGv hepatotoxicity.Pegvisomant (PEGv) is a growth hormone receptor antagonist approved for the treatment of acromegaly; one of its documented adverse effects is reversible elevation of hepatic enzymes. We report a 39-year-old male acromegalic patient with a pituitary macroadenoma who underwent transsphenoidal surgery. The patient's condition improved but GH and IGF-I levels did not normalize; as a consequence, we first administered dopamine agonists and then somatostatin receptor ligands (SRLs) with poor response. PEGv 15 mg every other day was added to lanreotide 120 mg monthly. The patient developed a severe hepatitis five months after starting the combination therapy. Elevated ferritin, iron, and transferrin saturation suggested probable hepatitis due to haemochromatosis. We performed a liver biopsy which showed an acute cholestatic hepatitis consistent with toxic etiology. A heterozygous genotype Acromegaly is a rare disease usually derived from a GH-secreting pituitary tumor.The increased mortality among persons with acromegaly includes higher prevalence of hypertension, hyperglycemia or overt diabetes, cardiomyopathy, and sleep apnea .Currently, surgery is the primary approach for treating most patients. If cure is not achieved with this treatment, pharmacotherapy should be considered. Somatostatin receptor ligands (SRLs) are usually the first line therapy for treating active acromegaly. Pegvisomant (PEGv), a pegylated GH receptor antagonist, is another effective therapeutic option that could be administered alone or in combination with SRLs –4. PEGv  UGT1A1⁎28 genotype associated with Gilbert's syndrome predicts an increased incidence of liver abnormalities during PEGv therapy in Spanish acromegalic patients [Bernabeu et al. found that thepatients .We report here clinical, biochemical, genotype, and histological findings in an Argentine acromegalic patient who developed a severe cholestatic hepatitis during PEGv therapy.In 1996 a 39-year-old man was referred to the Endocrinology Service of the Hospital Militar Central of Buenos Aires City because he presented arterial hypertension and acromegalic features. He had a two-year history of headaches, sweating, feet and hands enlargement, weight gain, erectile dysfunction, and hyperglycemia. His lab tests showed an elevated IGF-I level: >5.4 U/mL (NR: 0.6–5.4) and nonsuppressible GH postoral glucose tolerance test (OGTT) (GH nadir: 78 ng/mL). He underwent a magnetic resonance imaging (MRI) which showed a pituitary macroadenoma of 18 × 15 mm with suprasellar extension. The patient was operated on by transsphenoidal approach. The pathology report informed a diffuse eosinophilic pituitary adenoma. At that time we could not perform immunochemistry.The symptoms and hormonal test improved; however, GH and IGF-I levels were persistently elevated.In December 1996 bromocriptine 3.75 mg/day was indicated without normalization of IGF-I. Two years later, he was medicated with cabergoline 0.5 mg/week with poor tolerance and response; therefore, he returned to bromocriptine 5 mg/day. He stopped treatment and was lost to follow-up for five years. When the patient returned in 2003, an MRI showed no tumor but his IGF-I level was elevated; therefore, cabergoline was indicated. He was not consistent with the treatment because he presented poor tolerance to this drug. In 2005 he was started on octreotide 30 mg/month with partial response.Due to severe coronary heart disease, in October 2007 the patient underwent a coronary bypass surgery. In December 2008, as he presented glucose intolerance, a more sensible diet and metformin  were indicated.In 2010 a cholecystectomy was performed because of the development of gallstones. In 2011 two benign polyps and a villous adenoma were removed during a colonoscopy.In 2012 octreotide was switched to lanreotide 120 mg/month. Because of increased IGF-I levels, in March 2013 PEGv 15 mg every other day was added. Up to that moment the patient's liver function tests had always been normal. It is important to note that he did not drink alcohol. He was also medicated with atenolol 25 mg/day and enalapril 10 mg/day and continued metformin treatment. In May 2013 he presented transient symptoms of flu and a slight increase of transaminases, less than 3 times above the upper limit of normal (×ULN). In August 2013 the patient, under treatment with lanreotide and PEGv, had a normal IGF-I level, 154 ng/mL (81–225), but he unexpectedly experienced abdominal pain, severe asthenia, decreased appetite, and presented choluria the day before the visit to the hospital. Liver tests results showed elevated levels of aspartate aminotransferase (ASAT) 29.9 ×ULN, alanine aminotransferase (ALT) 45.3 ×ULN, alkaline phosphatase (APh) 2.26 ×ULN, and total bilirubin 2.5 ×ULN. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) were both normal (PT: 85% (70–120) and aPTT: 39 seconds (27–42)). Blood cell count was normal: hematocrit 45%, hemoglobin: 15 grams/dL, and white blood cell count: 7100 cells/mcL with no eosinophilia. There were no changes in the normal pattern of coagulation tests and blood cell count during the course of the hepatic disease. The tests for viral and autoimmune hepatitis were negative. Elevated ferritin, iron, and transferring saturation (TSAT) suggested probable hepatitis due to haemochromatosis. Liver and iron tests are shown in We performed a liver biopsy which showed an acute cholestatic hepatitis with confluent necrosis, which formed portocentral bridges of parenchymal collapse Figures . There wHaemochromatosis was also ruled out through a negative result of C282Y and H63D genotypes of haemochromatosis HFE. UGT1A1⁎28 associated with Gilbert's syndrome was performed and a heterozygous genotype UGT1A1⁎28 polymorphism was found. The UGT1A1⁎28 genotyping was performed by PCR amplification of a fragment of the UGT1A1 gene promoter, which included the TATA box, and Sanger sequencing of the amplification product.The genotypeLiver function tests returned to normal four months after PEGv discontinuation and they have been normal since then.Without combination therapy, IGF-I levels increased to 367 ng/mL (NR: 81–225) (1.63 ×ULN). In January 2014 lanreotide 120 mg monthly and cabergoline 0.5 mg/week were indicated with good tolerance and thus IGF-I levels normalized: 205 ng/mL.The association of PEGv therapy with drug-induced hepatic events is well-documented; however, the mechanisms responsible for its toxicity remain unclear .In the German Pegvisomant Observational Study, PEGv administration was associated with an increase in liver enzymes in 9% of patients . ACROSTU UGT1A1⁎28 genotype associated with Gilbert's syndrome predicts an increased incidence of liver abnormalities during PEGv therapy in Spanish patients. The excretion pathways of PEGv are unknown; in humans <0.6% of unchanged PEGv is excreted in urine. Nothing is known about the hepatic metabolism of PEGv and whether it requires UGT1A1-mediated glucuronidation [ UGT1A1⁎28 [According to Bernabeu et al., thenidation , 5. Alth1A1⁎28 .Drug-induced hepatotoxicity is a frequent cause of liver disease and can mimic all forms of acute and chronic hepatobiliary diseases. Based on ALT and APh levels, drug-induced liver injury is classified into acute hepatitis, cholestasis, or mixed patterns. This classification was established by the Council for International Organization of Medical Sciences and modified by US FDA Drug Hepatotoxicity Steering Committee . EstabliThe liver is the central organ responsible for the selective uptake, metabolism, and excretion of drugs, xenobiotics, and environmental toxins. This essential function predisposes the liver to drug toxicity .Adverse hepatic events caused by drugs can be considered to be either predictable (high incidence) or unpredictable (low incidence). The most common example of a drug causing predictable drug-induced liver injury (DILI) is acetaminophen. This type of drug injury has a short latency period, is dose-related, and is the most common form of DILI. On the contrary, idiosyncratic DILI is unpredictable, has longer/variable latency, and is less common .The pathogenesis of drug-induced liver injury usually involves the participation of a toxic drug or a metabolite that either elicits an immune response or directly affects the biochemistry of the cell. Most adverse drug-induced hepatic events are unpredictable and are either immune-mediated hypersensitivity reactions or idiosyncratic ones .The liver removes lipophilic chemicals from blood, including drugs, and biotransforms them into water-soluble metabolites which are excreted. This process involves cytochrome P450 (phase 1), conjugation (phase 2), and transport (phase 3). The expression of the enzymes and transporters involved in the hepatic handling of drugs are under the control of transcriptions factors (nuclear hormone receptors). In humans, polymorphisms of the genes that regulate each phase and transcription factors affect their activities and expression in response to environmental factors. So it is likely that the level of exposure to the toxic moiety (reactive metabolite or the parent drug) is the most upstream determinant of DILI .Acromegaly by itself is correlated with a high prevalence of gallbladder stones and the long-term treatment with SRLs increases the incidence of cholelithiasis . Our patLaboratory tests showed elevated ferritin and transferrin saturation suggesting probable hepatitis due to haemochromatosis, which was ruled out through a negative of C282Y and H63D genotypes and the liver biopsy. It is known that both ferritin and TSAT can also be elevated in many inflammatory conditions such as viral hepatitis, regular alcohol consumption, and fatty liver disease . UGT1A1⁎28 polymorphism [Filopanti et al. showed that in acromegalic patients treated with PEGv, multitherapies and previous episodes of liver disease were associated with increased risk of hepatotoxicity; however, they did not find any increased risk of liver toxicity in Italian patients withmorphism . Our pat UGT1A1⁎28 polymorphism in 54.2% of their patients; however, they could not confirm any association between this polymorphism and TET [Neggers et al. found only 13.5% of transiently elevated liver transaminases (TET) above 3 ×ULN, of which 83% occurred within the first year of combination treatment with somatostatin analogs and PEGv. In their study, they detected the and TET . Our pat UGT1A1⁎28 genotype in our Argentine patient might be related to his Spanish ancestry (he had three Spanish grandparents). This finding is in accordance with Bernabeu et al. study in Spanish acromegalic population. Besides there is another coincidence with their study: our patient is male [The presence of the is male .The mechanism of pegvisomant-induced liver injury remains the subject of conjecture. We report a patient with acromegaly who developed a severe drug-induced hepatitis during treatment with PEGv. When PEGv was discontinued, his laboratory tests normalized in four months.Although in this case we might assume that it was an idiosyncratic drug toxicity, to the best of our knowledge, it is the first case described in which its predominant clinical presentation resembles an acute cholestatic hepatitis associated with severe hemosiderosis, a different pattern of PEGv hepatotoxicity.


Aloysia gratissima, Baccharis dracunculifolia, Coriandrum sativum, Cyperus articulatus, and Lippia sidoides were proven to have strong antimicrobial activity on planktonic microorganisms; however, little is known about their effects on the morphology or viability of oral biofilms. Previously, we determined the EO/fractions with the best antimicrobial activity against Streptococcus mutans and Candida spp. In this report, we used a confocal analysis to investigate the effect of these EO and BF on the morphology of S. mutans biofilms  and on the metabolic viability of C. albicans biofilms. The analysis of intact treated S. mutans biofilms showed no statistical difference for thickness in all groups compared to the control. However, a significant reduction in the biovolume of extracellular polysaccharides and bacteria was observed for A. gratissima and L. sidoides groups, indicating that these BF disrupt biofilm integrity and may have created porosity in the biofilm. This phenomenon could potentially result in a weakened structure and affect biofilm dynamics. Finally, C. sativum EO drastically affected C. albicans viability when compared to the control. These results highlight the promising antimicrobial activity of these plant species and support future translational research on the treatment of dental caries and oral candidiasis.The essential oils (EO) and bioactive fractions (BF) from  Streptococcus mutans and Candida albicans are implicated in the establishment of dental caries and oral candidiasis, respectively, and constitute complex structures, often composed of interactive microorganisms which are organized for increased metabolic efficiency, resistance to stress, and virulence [ quorum sensing system [It has been long known that biofilms formed byirulence  with a sg system . S. mutans glycosyltransferases are the main constituents in the matrix of cariogenic biofilms and are recognized as critical virulence factors associated with dental caries [Extracellular polysaccharides (EPS) produced byl caries . An EPS-l caries . The EPSl caries .Biofilm formation on host tissues or medical devices mediated either by biotic (cell-surface proteins) or abiotic (surface hydrophobicity) factors also plays a role in the pathogenesis of yeast infections .Therefore, the development of specific-targeted therapies attenuating microbial virulence is of utmost importance and thus contributing to the management of dental caries and oral candidiasis, which have considerably affected the population worldwide , 7. With S. mutans [ Candida spp. [ Aloysia gratissima (fraction Ag4), Baccharis dracunculifolia (fraction Bd2), Coriandrum sativum (EO and fraction Cs4), Lippia sidoides (fraction Ls3), and Cyperus articulatus (EO). In general, Cs4 and Bd2 inhibited more than 90% of in vitro S. mutans biofilm formation at low concentrations (31.2 μg/mL), followed by Ag4 (62.5 μg/mL) and Ls3 (125 μg/mL) [ C. sativum and C. articulatus inhibited C. albicans biofilm from 62.5 μg/mL [μg/mL [In our previous studies, we screened twenty EO and their bioactive fractions and selected those with the most promising antimicrobial activity against. mutans  and Candida spp. , 11 as fL [μg/mL , respect S. mutans biofilms by assessing thickness, biovolume, and architecture and on the metabolic viability of C. albicans biofilms.Thus, based on these previous results and on the relevance of discovering new agents for the management of biofilm-dependent diseases, we investigated through a confocal analysis the effect of the EO and bioactive fractions earlier mentioned on the morphology ofhttp://webdrm.cpqba.unicamp.br/cpma/). The plant material was collected between November and January, during the morning. Voucher specimens were deposited in the herbarium of the Institute of Biology at the University of Campinas  and also registered in the herbarium of CPQBA, receiving identification numbers  at the Research Center for Chemistry, Biology, and Agriculture (CPQBA), University of Campinas (UNICAMP), SP, Brazil (available at  numbers .2SO4) to remove any trace of water, and filtered. This step is critical to absorb remaining moisture in the organic phase, resulting in water-free oil content. The solvent was finally evaporated to obtain the crude EO [The EO were obtained through hydrodistillation of the leaves or bulbs for 3 hours in a Clevenger-type system. The aqueous phase was extracted with dichloromethane and the organic layer was then isolated, dried with anhydrous sodium sulphate (Nacrude EO . The EO  A. gratissima (fraction Ag4), B. dracunculifolia (fraction Bd2), C. sativum (fraction Cs4 and EO), C. articulatus (EO), and L. sidoides (fraction Ls3).The EO were fractionated using the dry column method (cellulose column 2 cm × 20 cm) with Si gel 60  as the stationary phase and dichloromethane as the mobile phase, previously chosen by Thin Layer Chromatography. After elution, columns were separated into different parts for each EO based on polarity. The fractions obtained were chemically characterized by Thin Layer Chromatography and Gas Chromatography coupled to Mass Spectrometry (GC-MS) and tested for their antimicrobial activity. The selected EO and bioactive fractions showing the best antibacterial/antifungal effects . GC-MS analysis was performed using split injection (40 : 1), with the injector set at 220°C, column set at 60°C, with a heating ramp of 3°C/min and a final temperature of 240°C, and the MS detector set at 250°C. Helium was used as a carrier gas at 1 mL/min. The GC-MS electron ionization system was set at 70 eV. Samples of the bioactive fraction or crude oil were solubilized in ethyl acetate for the analysis. Then, retention indices (RIs) were determined by coinjection of hydrocarbon standards  and test samples under the same conditions. The oil components were identified by comparison with the data from the NIST 05 library, international literature, and by coinjection of authentic standards [Volatile constituents were identified using a Hewlett-Packard 6890 gas chromatograph coupled with an HP-5975 mass selective detector and HP-5 capillary column  and C. albicans CBS 562 were used.Reference strains of S. mutans was prepared using ultrafiltered  tryptone yeast extract  supplemented with 1% (w/v) glucose, incubated at 37°C, 5% CO2, overnight. This starter was used to prepare bacterial inoculum at midexponential growth phase for biofilm formation  .C. albicans was grown in yeast nitrogen base (YNB)  supplemented with 50 mM glucose. Fifty millimeters of medium was inoculated with yeast colonies from Sabouraud Dextrose Agar  plates, followed by incubation for 24 h at 37°C. Cells were harvested , washed twice with 0.15 M phosphate-buffered saline , resuspended in 40 mL of PBS, and read on spectrophotometer to have a concentration of 5 × 106 CFU/mL   immersed in 12-well flat-bottom cell culture plates  in triplicate. Human whole saliva was collected from two donors , clarified by centrifugation , sterilized and diluted (1 : 1) in adsorption buffer , and supplemented with the protease inhibitor phenylmethylsulfonyl-fluoride (PMSF) at a final concentration of 1 mmol/L. The glass slides were placed as much vertically as possible in 12-well plates and inoculated with S. mutans suspension in buffered ultrafiltered UFTYE medium supplemented with 1% (w/v) sucrose and incubated at 37°C, 5% CO2. S. mutans biofilms were grown undisturbed during 19 h, and later the culture medium was replaced daily until the end of the experimental period, which totalized 72 h  with the bioactive fractions or vehicle (propylene glycol) at the given concentrations . To asstrations , both di C. albicans biofilms, glass slides (324 mm2 surface area) were initially immersed in 12-well flat-bottom cell culture plates  containing fetal bovine serum (FBS)  and incubated aerobically for 24 h at 37°C (pretreatment phase). The slides were then washed with PBS to remove residual FBS, moved to new plates containing a standardized C. albicans suspension (5 × 106 CFU/mL) and incubated aerobically for 90 minutes at 37°C (adhesion phase). The slides were gently transferred to new plates to ensure the removal of nonadhered yeasts and were finally immersed in YNB medium containing the EO (μg/mL (MIC); Sigma-Aldrich, St. Louis, MO, USA), in triplicate. The plates were incubated aerobically at 37°C for 48 h (biofilm formation phase) .μg/mL or 15.7 to 125 μg/mL were tested at 600 μg/mL and 800 μg/mL, respectively, in view of the higher microbial resistance observed in biofilm cultures [The sample concentrations used in this study were based on our previous findings of antimicrobial susceptibility for the bioactive fractions and EO , 11. Samcultures .μm intervals) at each of the positions [μm3·μm−2), average thickness (μm), and biofilm coverage (μm) on the slide surface.Extracellular polysaccharides (EPS) were labeled via incorporation of Alexa Fluor 647 dextran conjugate  (absorbance/fluorescence emission maxima of 647/668 nm), while bacterial cells were stained with SYTO 9 (485/498 nm)  30 minutes before confocal imaging. The analysis of intact biofilms was performed using a Zeiss LSM 780-NLO confocal laser scanning microscope  equipped with a EC Plan-Neofluar 63x oil immersion objective lens (excitation wavelength 810 nm). Each biofilm was scanned at least at 5 randomly selected points and a confocal image series was generated by optical sectioning   and 15 μL of Concanavalin A, Alexa Fluor 488 Conjugate (from a 5-mg/mL stock)  were mixed into 3 mL of PBS to give final concentrations of 10 μM and 25 μg/mL, respectively, in the wells [Following formation and treatment ofThe data were analyzed qualitatively based on the morphology and architecture of EPS and bacterial cells and quantitatively by one-way analysis of variance (ANOVA) followed by Dunnett's multiple comparison test  with type I error set as 0.05. 2D and 3D images were generated and processed using ZEN 2012 Black Edition ©Carl Zeiss Microscopy Gmbh platform. All experiments were performed in triplicate.The medicinal aromatic plants were obtained exclusively from a germoplasm bank  and subm4, trans-nerolidol in Bd2, thymol in Ls3, 1-decanol in Cs4, decanal in C. sativum EO, and α-pinene in C. articulatus EO. These compounds alone were proven to have antimicrobial activity against gram-positive bacteria [The chemical composition of the EO and selected bioactive fractions can be seen in bacteria  and yeasbacteria , 20 thatThe fractionation process adopted in this bioguided study is well established in the literature , 22. As Confocal laser scanning microscopy is a useful tool for studying morphology of bacterial biofilms , as it a S. mutans biofilms. Overall, confocal imaging revealed that most experimental samples at the given concentrations produced both qualitative (P > 0.05), as seen in To the best of our knowledge, this is the first confocal study in the literature determining the effect of these bioactive fractions from EO on the biovolume and architecture oflitative  and quanlitative  alterati S. mutans biofilms treated with the fractions. The biovolume provides an estimation of biofilm biomass in relation to the substratum area [4 and Ls3 (P < 0.05) according to the COMSTAT analysis. The groups Bd2 and Cs4 showed reduced levels of bacterial volume but not of EPS. Therefore, our findings support the view that the bioactive fractions of Ag4 and Ls3 disrupt biofilm integrity and may have created porosity in the biofilm, as there was no change in thickness but biomass of EPS and bacteria was significantly reduced. This phenomenon could potentially result in a weakened structure and affect the biofilm dynamics, and this hypothesis may be a platform for the development of new antimicrobial targets on biofilm-dependent oral diseases such as caries, candidiasis, and corelated ailments.tum area . In our An association has been established between biofilm formation and intrinsic resistance to antimicrobial stress , which e A. gratissima and L. sidoides fractions had significant effects on bacterial viability acting as bactericides [ S. mutans pathogenicity, that is, production of EPS [As EO are composed of numerous chemical compounds, their antimicrobial activity might be attributed to several distinct mechanisms . Brieflyericides  and alson of EPS .Due to their hydrophobicity, EO molecules are able to pass through the cell wall and penetrate fatty acid chains of the phospholipid bilayer, rendering the cell membrane more permeable and causing leakage of intracellular contents. Such loss of cell homeostasis can culminate in lysis and cell death . This me A. gratissima and L. sidoides reduced its amount, indicating that one of their putative mechanisms of action might be related to the inhibition of glycosyltransferase activity.EPS are an outstanding key-factor in biofilm formation being produced by glycosyltransferases . The fraOur coverage (EPS/bacteria) data  demonstr C. sativum [ C. articulatus [Candida activity. We reported that C. sativum EO, rich in decanal and trans-2-decenal (major compounds), has strong fungicidal effects against Candida albicans and non-albicans and acts by binding to membrane ergosterol, which increases ionic permeability and leads to cell death. This is the same mechanism of action of polyenes such as nystatin and amphotericin B [ C. articulatus presents antifungal properties due to constituents such as α-pinene (major compound) that could be able to destroy cell integrity and inhibit respiration and the ion transport processes, leading to cell death [In previous studies, sativum  and C. aiculatus  have stoericin B . C. artill death . C. sativum and C. articulatus EO inhibited yeast biofilm adherence onto a polystyrene substrate from 62.5 μg/mL [μg/mL [ Candida spp. than their fractions either on planktonic or biofilm cultures [Both.5 μg/mL  and 1.95L [μg/mL , respectcultures , 12; tha C. sativum EO ( C. sativum EO emerges as a promising candidate for nonclinical and clinical toxicology testing for the development of new drugs to treat denture-related oral candidiasis.In our study, confocal microscopy revealed that both OE altered the viability of yeast cells compared to the vehicle , most notivum EO , which s C. sativum EO (The treatment withtivum EO  affectedtivum EO . This fi A. gratissima and L. sidoides significantly reduced the biovolume of extracellular polysaccharides and bacterial cells in the S. mutans biofilm model used, but there was no difference with regard to biofilm thickness. Taken together, our findings support the view that these fractions disrupt biofilm integrity and may have created porosity in the biofilm, as biomass decreased but thickness was unaltered. Furthermore, C. sativum EO drastically affected the viability of C. albicans cells. These results highlight the promising antimicrobial activity of these plant species and suggest avenues for future translational research on the treatment of dental caries and oral candidiasis.The bioactive fractions from


Gongronema latifolium fruit has wide application in ethnomedicine, especially in maintaining healthy living and general body healing. We therefore investigated the antioxidant, immunomodulatory activities, and safety of its ethanol extract and fractions. The in vitro antioxidant activities of the extract and fractions were determined using 2,2-diphenyl-1-picrylhydrazyl (DPPH) test while in vivo activities were determined using carbon tetrachloride (CCL4) induced oxidative stress. Cell and humoral mediated immune responses were also evaluated together with toxicity studies. The extract, ethyl acetate, and methanol fractions showed inhibition of DPPH radical with IC50s 120, 90, and 60 μg/mL, respectively. Methanol fraction at 200 mg/kg produced significant (P < 0.05) inhibition of lipid peroxidation (MDA conc. 1.2 μmol/L) compared to control (2.8 μmol/L). Both ethyl acetate and methanol fractions at 200 mg/kg produced significant (P < 0.05) phagocytic index of 0.021 and 0.025, respectively, compared with control (0.01). Significant (P < 0.05) elevations of white blood cells, aspartate aminotransferase, alanine aminotransferase, and alkaline phosphatase were noticed on the 91st day at higher doses. Generally, this study justified the traditional use of G. latifolium fruit for general body healing and maintenance of healthy living. Long term administration is safe on the haematological and biochemical systems especially at lower doses and its toxicity at higher doses is reversible. The human environment together with the biological processes contributes significantly to free radical production . When thGongronema latifolium Benth (Asclepiadaceae) is a climber that is widely distributed in tropical Africa. It is commonly known as utazi in South Eastern Nigeria while in Ghana, Senegal, and Sierra Leone, it is called akan-asantes, gasub, and ndondo-polole, respectively [ G. latifolium fruit.ectively . Its leaectively . The ethectively . Also, tectively . The hypectively –10. The ectively . In Sout G. latifolium were collected from Nsukka, Enugu State, Nigeria, in March, 2012 and authenticated by a taxonomist, Mr. Alfred Ozioko of Bioresource Development and Conservation Project, Nsukka, Enugu State, Nigeria. The fruits were subsequently cleaned, air-dried under room temperature for 20 days, and pulverized.The fruits ofn-hexane, chloroform, ethyl acetate, methanol, hydrogen peroxide, hydrochloric acid, ethanol, DPPH , epinephrine, trichloroacetic acid, thiobarbituric acid, ovalbumin, and tetanus toxoid were all obtained from Sigma Chemicals CO, USA. All laboratory reagents including distilled water were freshly prepared when required.Analytical grades of  in vacuo using rotary evaporator at 40°C. The extract (116 g) was adsorbed on silica gel and eluted in succession with n-hexane, chloroform, ethyl acetate, and methanol. The eluent was filtered and concentrated in vacuo using rotary evaporator at 40°C to obtain the n-hexane, chloroform, ethyl acetate, and methanol fractions. All the extracts and fractions were stored in the refrigerator between 0 and 4°C until used.About 3 kg of the pulverized fruit was cold-macerated in aqueous ethanol (70%) for one week. The resulting solution was filtered using Whatman filter paper. The filtrate was concentrated to drynessThe phytochemical analyses of the fruit extract and fractions were carried out using standard methods , 13. QuaSwiss male and female albino rats 130 ± 7 g and mice 30 ± 4 g (3 months old) were employed for the study. All the animals were obtained from the animal house of the Department of Pharmacology and Toxicology, Nnamdi Azikiwe University, Agulu campus. The animals were housed in standard laboratory conditions of 12 h light, room temperature, and 40–60% relative humidity and fed with rodent feed (Guinea Feeds Nigeria Ltd.). They were allowed free access to food and water.Albino rats were used for the antioxidant studies while mice were used for immunomodulatory studies. In both studies, the animals were divided into 8 groups of 5 animals each. Groups 1 and 2 received ethanol extract while groups 3 and 4 received ethylactate fraction and groups 5 and 6 methanol fraction. The extracts and fractions were administered orally at the doses of 200 and 400 mg/kg. Groups 7 and 8 served as the control and received 200 mg/kg of standard (vitamin C or Noni capsule) and 10% tween 80, respectively.For the chronic toxicity studies, eighty male rats were randomly divided into four groups  of twenty rats per group. The extract was administered orally at doses 750, 1500, and 3000 mg/kg to groups A, B, and C once daily for 90 days. The control, group D, received 10 mL/kg of normal saline. Physical observation of the animals was done on daily basis while their weights were taken every week.All animal experiments were conducted in accordance with NIH guide for the care and use of laboratory animals  and appr50 was categorised based on this guideline [Acute toxicity study was carried out according to OECD guideline for acute oral toxicity testing. A total of six mice and rats were subjected to limit test at 5000 mg/kg BW. Three animals each were administered this dose (p.o.) and observed for 48 hours for mortality or obvious toxic symptoms and thereafter for 14 days. This was followed by three additional animals each at the same dose. The LDuideline .μg/mL) of the extract and fractions. The final volume of the resulting solution was adjusted to 5 mL using methanol. The absorbance of the mixtures was measured at 520 nm after incubation in the dark for 30 minutes at room temperature. Ascorbic acid was used as standard.The DPPH test was evaluated using the method of Patel and Patel . DPPH so DPPH scavenging activity = 100{(AC − AS)/AC}. AC = absorbance of negative control. AS = absorbance of Sample.The absorbance of the test substances and the negative control (0.5 mL of DPPH solution and 4.5 mL of methanol) was compared and the value used as parameter for the evaluation of antioxidant property. The free radical scavenging activity was obtained using the relationship shown below.4, 1.5 mL/kg BW. Blood samples were collected from the animals through retroorbital plexus 8 h after CCL4 administration. The serum was used for the determination of antioxidant enzyme activity  and lipid peroxidation according to the method described by Ogbunugafor et al. [The animals were anesthetized with light ether anaesthesia and blood samples (2 mL) were collected from the animals through retroorbital plexus into plain tubes for the pretreatment basal serum antioxidant assay. After the blood collection, the animals were treated with the extract and fractions once per day for 21 days. The animals were then given the acute oral dose of CCLr et al. . SuperoxμL of each of the blood samples was mixed with 3 mL of sodium carbonate and optical density was measured spectrophotometrically at 650 nm. The phagocytic index (K) was calculated as follows [1 and OD2 are optical densities at t1 and t2. t1 and t2 are time intervals at 0 and 15 minutes.The phagocytic index of the extract and fractions was determined using carbon clearance test. The animals were treated with the extract and fractions for 14 days. After 3 hrs posttreatment on the 14th day, the animals were given intravenous injection through the tail vain of 1 mL/100 g of Indian ink. Blood samples were drawn through retroorbital plexus into heparinised EDTA tubes at 0 and 15 minutes after injection. 25  follows :(1)K=inThis was evaluated using the method of Makare et al. . On the Following 14th day of daily treatment, blood samples were collected through retroorbital plexus into heparinised EDTA tubes and analysed for total (TLC) and differential leucocyte counts (DLC) by fixing blood smear and staining with Leishman's stain.μg/mL ovalbumin or tetanus toxoid on the 5th and 14th days. Blood samples were collected through retroorbital plexus on the 14th and 21st days for determination of primary and secondary antibody responses, respectively. The antisera were separated and used for the estimation of IgG1 and IgG2a using ELISA as described by Duddukuri et al. [For the ovalbumin (OVA) and tetanus toxoid immunization, the animals were intraperitoneally injected with 10 i et al. .Five rats from each group were anaesthetized using light ether anaesthesia on the 31st, 61st, and 91st days of the study. Blood samples were collected through retroorbital puncture and heparinised whole blood samples were used for the estimation of haemoglobin (Hb), packed cell volume (PCV), red blood cell (RBC), and white blood cell (WBC) counts using the method described by Kelly . For theP < 0.05 and P < 0.01.The results were analysed using SPSS version 18 and presented as mean ± SEM. Mean comparison was determined using one-way ANOVA and turkey test. Differences between control and extract treated groups were considered significant at  G. latifolium fruit showed the presence of various chemical constituents  increase in serum catalase activity that is higher than that of vitamin C (P < 0.05) increase in serum superoxide dismutase enzyme just as vitamin C (P < 0.05) reduction in lipid peroxidation compared with the control (Ethyl acetate and methanol fractions produced significant (itamin C  while etitamin C . Methano control .P < 0.05) increase in phagocytic index while, in the delayed type hypersensitivity reaction, the extract and fractions at 200 and 400 mg/kg BW produced significant increase in paw thickness when compared with negative control. Noni capsule (50 mg/kg) also showed significant (P < 0.05) phagocytic index and delayed type hypersensitivity reactions (The extract at 400 mg/kg BW and the fractions at the tested doses increased clearance of carbon particles from the blood as indicated by significant (eactions .P < 0.05) elevation of neutrophils ; however only ethyl acetate fraction at 400 mg/kg showed differential significant (trophils .P < 0.05) stimulation of IgG1 and IgG2a just as the standard (Noni capsule) at 50 mg/kg BW against both ovalbumin and tetanus toxoid (P < 0.05) production of IgG1 and IgG2a  alteration of the body weights (P < 0.05) elevation of WBC count , superoxide dismutase (SOD) enzymes, and decreased malondialdehyde (MDA). SOD converts superoxide radicals to hydrogen peroxide, while CAT converts the hydrogen peroxide to water and oxygen [Exposure of the animals tod oxygen . Reactivd oxygen . Therefod oxygen , 8 usingd oxygen  reported4 leading to reduced free radical formation and cellular damage. The potent antioxidant activity of flavonoids, their ability to scavenge hydroxyl radicals, may be the most important function of flavonoids and underlies many of their actions in the body [ G. latifolium leaves has been found to inhibit food deterioration through inhibition of oxidative enzymes such as lipoxygenase [ G. latifolium fruit may be mediated by more than a single mechanism.Phenolic compounds have been noted to induce the activation of multidrug resistant protein gene . This prthe body . They dothe body . Their cthe body . Saponinthe body . Tannin xygenase . Followixygenase , it is mP < 0.05) enhanced cellular immunity (increased phagocytic index), increased delayed hypersensitivity reaction (increased SRBC induced hypersensitivity reaction), increased innate immunity (increase in leukocyte count), and increased stimulation of the mouse IgG1 and IgG2a. These indicate that extracts of G. latifolium can boost immunity. Free radicals weaken the immune system [The extract and fractions significantly  reduction of PCV, Hb concentration, and RBC count is an indication of unlikeliness of the extract to cause anaemia [The acute toxicity studies revealed that the extract did not cause any death in both mice and rats up to 5000 mg/kg. The nonsignificant ( anaemia .P < 0.05) elevation of ALT, AST, and ALP was noticeable at higher doses. Histological examination of the liver showed that the extract at 3000 mg/kg produced vacuolar degeneration at the 91st day which however showed convincing signs of reversibility after 28-day posttreatment. Vacuolar changes usually occur due to overaccumulation of its storage products or waste products and can also occur secondarily due to endocrine disorder or increased intake of steroids [AST, ALT, and ALP are liver marker enzymes. Their increase in serum is roughly proportional to the extent of liver damage . The sigsteroids . This efsteroids . G. latifolium fruit extract as an antioxidant and immunostimulator in rats. Although we are much encouraged by these effects of the extract in our rat model, further study of the mutagenic and teratogenic effects is needed before the use of G. latifolium fruit extract in humans is justified.Our findings confirm the value of G. latifolium fruit have antioxidant and immunostimulant effects. This justifies the folkloric use in general body healing and maintenance of healthy living. Its long term administration is safe especially at lower doses. However, its liver toxicity at high dose is reversible.The extracts and fractions of


To determine the optimal vital sign predictor of adverse maternal outcomes in women with hypovolemic shock secondary to obstetric hemorrhage and to develop thresholds for referral/intensive monitoring and need for urgent intervention to inform a vital sign alert device for low-resource settings.or severe end organ dysfunction morbidity); and (3) a combined severe maternal and critical interventions outcome comprising death, severe end organ dysfunction morbidity, intensive care admission, blood transfusion ≥ 5 units, or emergency hysterectomy. Two threshold parameters with optimal rule-in and rule-out characteristics were selected based on sensitivities, specificities, and positive and negative predictive values.We conducted secondary analyses of a dataset of pregnant/postpartum women with hypovolemic shock in low-resource settings (n = 958). Using receiver-operating curve analysis, we evaluated the predictive ability of pulse, systolic blood pressure, diastolic blood pressure, shock index, mean arterial pressure, and pulse pressure for three adverse maternal outcomes: (1) death, (2) severe maternal outcome , diastolic blood pressure and pulse pressure for severe maternal outcome (p<0.01), and systolic and diastolic blood pressure, mean arterial pressure and pulse pressure for severe maternal outcome and critical interventions (p<0.01). A shock index threshold of ≥ 0.9 maintained high sensitivity (100.0) with clinical practicality, ≥ 1.4 balanced specificity (range 70.0–74.8) with negative predictive value (range 93.2–99.2), and ≥ 1.7 further improved specificity (range 80.7–90.8) without compromising negative predictive value (range 88.8–98.5).For women with hypovolemic shock from obstetric hemorrhage, shock index was consistently a strong predictor of all adverse outcomes. In lower-level facilities in low resource settings, we recommend a shock index threshold of ≥ 0.9 indicating need for referral, ≥ 1.4 indicating urgent need for intervention in tertiary facilities and ≥ 1.7 indicating high chance of adverse outcome. The vital sign alert device incorporated values 0.9 and 1.7; however, all thresholds will be prospectively validated and clinical pathways for action appropriate to setting established prior to clinical implementation. Approximately 6% of deliveries are complicated by obstetric hemorrhage . DespiteVisual estimation of blood loss is routinely used to assess severity and guide resuscitation; however, blood loss is often underestimated . Vital sWithin the general population, shock index (SI), the ratio of pulse to SBP, is proposed as an earlier marker of hemodynamic compromise than conventional vital signs . Trauma We sought to determine the optimal vital sign predictor for severe adverse maternal outcomes among women in hypovolemic shock secondary to obstetric hemorrhage, and to develop thresholds corresponding to early detection triggers for immediate referral or intensive treatment for incorporation into a semi-automated handheld, vital sign alert (VSA) device –18 whichWe compiled data from 967 women comprising pre-intervention/control participants from four studies conducted by the Safe Motherhood Program at the University of California, San Francisco that evaluated the effectiveness of the non-pneumatic anti-shock garment (NASG) to reduce adverse maternal outcomes for women with hypovolemic shock secondary to severe obstetric hemorrhage: Egypt 2004 (n = 158), Egypt 2006–2008 (n = 432), Nigeria 2004–2007 (n = 182) and Zambia and Zimbabwe 2007–2012 n = 195). Detailed descriptions of these studies have been published elsewhere 5. Detail. Three oWe excluded 1 individual who participated in the Zambia trial but was lost to follow-up and had no outcome data for death, and 8 individuals across all studies that had no vital sign data recorded or whose vital signs were indicated as non-palpable. Participants missing other predictors or outcomes were retained, resulting in a final analytic sample of 958 participants.The data analyzed were based on data captured from the parent studies in which, upon enrollment, patients’ vital signs, level of consciousness, urine output, blood loss, IV fluids, blood products transfused and uterotonics administered were recorded at 15 minute intervals, until the cause of bleeding was identified and treated, vital signs were stable  for at least two hours, and blood loss had decreased to approximately 25–50 mL per hour. Predictor variables for this analysis were values at the measurement interval with the highest SI (pulse/SBP) within the first hour after study entry. Variables included pulse, SBP, diastolic blood pressure (DBP), mean arterial pressure (MAP = (2 x DBP + SBP)/3), SI (pulse/SBP), and pulse pressure (SBP-DBP). Severe shock at study entry was defined as MAP less than 60 mmHg, below which perfusion of vital organs has been proposed to be inadequate –25. BP wOutcomes comprised any severe adverse maternal event related to obstetric hemorrhage, and included organ system dysfunction-based criteria and intervention-based criteria. Although the outcomes for the original trials were determined prior to the development of the WHO “Near-Miss” criteria , they arWe first evaluated the area under the curve (AUC) and associated 95% confidence interval for each of the predictors with each of the maternal outcomes using non-parametric receiver-operating curve (ROC) analysis . We thenDemographics and participants’ status at the time of study entry are presented in Distributions of vital signs at study entry are presented in The proportion of women with SI greater than each of the thresholds evaluated is as follows: SI ≥ 0.7: 99.6%, SI ≥ 0.9: 95.0%, SI ≥ 1.4: 32.3% and SI ≥ 1.7: 13.8% (not shown). The performance of SI ≥ 0.7, the upper limit of normal SI for the non-pregnant population , and SI Our analysis found SI to be the most consistent predictor across a selection of severe maternal outcomes and critical clinical indicators in women with hypovolemic shock secondary to obstetric hemorrhage. High sensitivity combined with clinically practical specificity drove our choice of SI ≥ 0.9 as the threshold for referral to a tertiary facility capable of comprehensive emergency obstetric care or rigorous monitoring within such a context. We chose a higher threshold of SI ≥ 1.4 to indicate need for intensive intervention because this value maximized identification of women needing rapid resuscitation balanced with high sensitivity and negative predictive value. However, for the purposes of clinical validation of the VSA device, an urgent resuscitation threshold of SI ≥ 1.7 was implemented in order reduce the rate of false positives and loss of trust in the VSA device at its inception; however all 3 thresholds will be evaluated prospectively. Such a validation process will help to further refine the higher action threshold, as it may vary based on clinical context and population.Previous research has suggested a normal SI range of 0.7–0.9 for obstetric populations, with 0.9 representing the transition into abnormality . ResearcThe literature on SI threshold indicating highest risk of adverse outcome is sparse. Trauma-related mortality has been found to be significantly increased above SI 1.0, 1.4 and 1.8 . For posThis study represents a preliminary evaluation of SI as a predictor of adverse maternal outcomes in women with obstetric hemorrhage/hypovolemic shock in low-resource settings. We draw upon a large sample of women with a variety of obstetric hemorrhage etiologies, and prioritize the most robust and salient outcome of mortality. However, the generalizability of our findings is limited to similar clinical and population contexts, and certain characteristics of our data and analysis represent limitations to our study, which may affect the interpretation of our results. First, all women in the original studies were experiencing hypovolemic shock secondary to severe obstetric hemorrhage; therefore, they already met the WHO definition of “near-miss” . Second,The developing evidence base on the utility of SI, strengthened by the results from our analysis, indicates a potential role for SI in early diagnosis and management of shock and for reducing adverse outcomes in obstetric populations. We suggest a lower SI threshold of 0.9 indicating need for referral to tertiary facility or rigorous monitoring within tertiary care. Based on our results, we suggest higher SI thresholds of 1.4 to indicate the urgent need for intensive treatment, and 1.7 as indicative of high risk of adverse event. Further research should evaluate these thresholds prospectively and focus on implementing SI as a tool at multiple clinical levels with different categories of care providers to maximize its utility within clinical obstetric early warning systems , 18.