Hepatocyte Growth Factor (HGF) enhances cytotoxicity of paclitaxel (PTX) and cisplatin (CDDP) in human ovarian cancer cells. Because of potential pitfalls of HGF exogenous administration, we investigated whether HGF serum concentration might be alternatively raised in vivo by administering low molecular weight heparin (LMWH).The main HGF pharmacokinetic parameters were evaluated following acute and chronic LMWH treatment. First, women, operated on for gynaecological tumors, were treated with a single dose of calcium nadroparin and studied for 12 hours. Next, women operated on for benign or malignant gynaecological tumors were treated daily with calcic nadroparin for one month. Subsequently, the biological activity of the measured HGF serum levels was tested in assays of ovarian cancer cell sensitization to drugs.ss, Cmax and Caverage were about four-fold that of the control group, whereas Cmin was three-fold. In the patients treated chronically median HGF serum levels rose about six-fold in the first week, and decreased but remained significantly higher after one month. The pharmacokinetic of nadroparin-dependent HGF increase were similar in the two groups. The HGF concentrations measured after both acute and chronic treatment were found to be effective in sensitising ovarian cancer cells to chemotherapeutics.In the short-term treated group, median HGF AUCThis study raises the possibility of using LMWH to increase HGF serum concentration and to take advantage of its biological activities. In particular, nadroparin might be used as a chemo-potentiating agent in epithelial cell ovarian carcinoma through its action on HGF serum concentration.ClinicalTrials.gov ID: NCT01523652 Similarly, HGF could be used to facilitate and accelerate regeneration after acute and chronic renal injury [.The biological activities of Hepatocyte Growth Factor have been thoroughly investigated both in vitro and in vivo. HGF is implicated in epithelial–to-mesenchymal transition, thus promoting migration, invasion and motility of several cellular types . It prom injury ,8. After injury .http://www.vai.org/met/). In line with these findings, inhibitors of the HGF-MET axis have been proposed for cancer treatments and are presently undergoing clinical trials. In a preclinical model of ovarian cancer, an orally available small-molecule inhibitor of c-Met, PF-2341066, has been reported to reduce tumour burden and metastasis [Several studies have also pointed out the importance of the paracrine action of HGF on neoplastic transformation in several tumor types, such as in the Epithelial Cell Ovarian Carcinoma (ECOC) . The intastasis .However, in 2004, Rasola A. et al. unexpectedly revealed that pre-treatment with HGF enhances the apoptotic response of human ovarian cancer cells to very low doses of paclitaxel (PTX) and cisplatin (CDDP) . ThroughFollowing these results, we investigated how the HGF concentration might be raised in vivo. Although, at least hypothetically, it could be possible to use gene therapy or to administer human recombinant HGF (hrHGF), the latter therapies are not standardised and require appropriate studies. We consider an alternative way of raising HGF serum concentration by administering low molecular weight heparin (LMWH) as suggested by preliminary data in the literature -19, takiThe study consisted of two phases. In the first phase, the main HGF pharmacokinetic parameters were evaluated, comparing a group of six women treated with a single dose of calcic nadroparin to a control group of six untreated women. Venous blood was drawn in both groups at 0, 30, 60, 90, 120, 150, 180, 240, 300, 360, 480 and 720 min. In the second phase, the HGF basal and maximum concentrations were measured in 17 women, undergoing one month of calcic nadroparin daily treatment. Venous blood was drawn twice on day 1 (at 0 and 90 min after nadroparin administration), then once on days 8 and 28 (at 90 min after LMWH injection). Calcic nadroparin was given subcutaneously at 2850 IU/0.3 ml anti-Xa .2. In the control group, four were healthy women volunteers and two patients submitted to gynaecological pelvic surgery, but these women were not treated with prophylactic LMWH. Their median age was 30 years (range 25–60), and their median BMI was 19.1 kg/m2.In the first phase, 12 patients were enrolled, 6 treated with nadroparin for prophylactic anticoagulation and another 6 untreated as the control group. The six nadroparin-group patients were affected by benign pelvic gynaecologic diseases: three requiring laparoscopy and three laparotomy. All of them were treated at the Gynaecological Oncology Unit of the hospital Azienda Sanitaria Ospedaliera Ordine Mauriziano in Turin, Italy. Their median age was 42 years (range 35–52), and their median BMI was 22.8 kg/m2.In the second phase, 17 patients were enrolled among women planning gynaecological pelvic surgery and treated for 4 weeks with nadroparin for prophylactic anticoagulation. All these patients underwent laparotomy; ten were affected by malignancy (ECOC) and seven by benign  pelvic gynaecologic diseases. Their median age was 53 years (range 41–75 years), and their median BMI was 22.1 kg/m-1, platelets ≥ 150000 μl-1, creatinine 0.6-1.2 mg/dl, total bilirubin ≤ 1 mg/dl, AST ≤ 35 IU/l, and ALT ≤ 45 IU/l.Inclusion criteria for phase 1 were as follows: age ≥ 18 years, ECOG Performance Status ≤ 1; neutrophils ≥ 1500 μlInclusion criteria for phase 2 were the same as for phase one, together with moderate-high risk of deep venous thrombosis (DVT), anticipated anaesthesia > 30 min, obesity, varicose veins, previous DVT and/or pulmonary embolism, thrombophilia, prolonged immobility, and congestive heart failure.General exclusion criteria were as follows: serious liver or renal diseases, diabetes, hyperlipidemia, serious osteoporosis, pregnancy or lactation, high risk of bleeding , inflammatory or infectious diseases, previous cancer, consumption of coffee, tobacco or ethanol in the last 24 hours, and women in treatment with immunosuppressive drugs, contraceptives, lipid lowering agents, or antiaggregants.All patients and healthy volunteers gave written informed consent prior to enrolment into the study, which was approved by the local ethics committee .Cisplatin (CDDP) was from Bristol-Myers Squibb ; APC conjugated Annexin V was from Boehringer Mannheim  and tetramethylrhodamine methyl ester (TMRM) was from Molecular Probes ; all of the other chemicals were from Sigma . Pure human recombinant HGF was from R&D Systems . SK-OV-3 cells were purchased from American Type Culture Collection  and grown as suggested by the provider.The ELISA kit for the determination of HGF in patients’ serum was purchased from BioSource . Blood samples were collected using BD Vacutainer® tubes without additives, blood for serum preparation was left to coagulate and then centrifuged at 3000 rpm for 10 minutes. All samples were coded, stored at −65°C and analysed within 2 months.Apoptosis assay was used to evaluate if the HGF concentrations measured in patients after nadroparin administration were effective in sensitising ovarian cancer cells to CDDP. SK-OV-3 ovarian cancer cells were pre-treated with decreasing concentrations of recombinant HGF and, after 48 hours, exposed to medium supplemented with 20 μM CDDP to induce apoptosis. We then measured the percentage of live cells, i.e. cells that did not display either early or late apoptotic features. Flow cytometry recordings of several independent apoptotic changes were performed by a single-tube analysis as previously described . Briefly, after treatment, cells were resuspended in HEPES buffer  and incubated for 60 min at 4°C in APC-conjugated Annexin V, TMRM (200 nM), and propidium iodide  to detect phosphatidylserine exposure on the cell surface, mitochondrial inner membrane electrochemical gradient, and plasma membrane integrity, respectively. Cell morphology changes were analysed following variations of the forward and side light scatter. Samples were acquired on a CyAn flow cytometer and analysed with Summit V4.3 software .max (maximum concentration), Cmin (minimum concentration), Caverage (average concentration at steady state), Tmax (time to reach maximum concentration) and AUCss (area under concentration/time curve at steady state).The pharmacokinetic study entailed a typical model-free approach, the non-compartmental analysis (NCA). The goal of NCA is to provide an estimate of the main pharmacokinetic parameters by the trapezoidal rule, the only assumption being that the terminal elimination phase can be described by a mono-exponential equation, following first order kinetics. NCA was performed using Kinetica 2000 4.1.1 , estimating the following HGF parameters: CHGF pharmacokinetic parameters were compared during phase 1 (control vs. nadroparin group), phase 1 vs. 2 (nadroparin group) and phase 2  by the Mann–Whitney test, while the four HGF levels during phase 2 were compared by repeated measures analysis of variance plus within-subjects contrasts. All reported p values were obtained by two-sided exact method, at the conventional 5% significance level. Data were analysed as of March 2009 by SPSS 17.0 . All data are presented as median values and range.The effect of a single nadroparin administration on HGF serum levels was investigated during phase 1 (control vs. nadroparin group); two different HGF kinetics profiles can be recognised at a glance, corresponding to control untreated women and LMWH-treated patients , affecting its kinetic behaviour and steady state profile (AUCss from 534 to 2157 ng*min/ml). The median Tmax in LMWH group (93 min) can be used as a reference in phase 2 to verify if the HGF concentration’s increase after a single LMWH administration persists even after a month of daily repeated treatment with nadroparin.In the LMWH group, median HGF AUCmax at day 0, 90 min after LMWH administration). Median HGF Cmax was almost constant at day 8 (4.19 ng/ml) after the first week of LMWH treatment, whereas at day 28, levels decreased to 2.96 ng/ml; this trend is represented in Figure The 17 patients taking part in phase 2  were all treated with daily nadroparin for 1 month. Median HGF serum levels went up from 0.65 ng/ml  to 4.59 ng/ml (Cmax in phase 1 vs. 2 (both obtained after a single nadroparin administration), the effect on HGF maximum concentrations seems to be minor, even if the statistical significance is borderline (Table From testing HGF Cmax concentrations after the first nadroparin administration (HGF post_1), after 8 and 28 days of treatment (HGF post_2 and HGF post_3) are represented in Table Basal HGF levels before nadroparin administration (HGF_pre) and HGF CAnalysing the phase 2 HGF concentrations, it is possible to confirm the HGF pharmacokinetic nadroparin-dependent alteration, already found in phase 1. Using a repeated measures analysis of variance model, the HGF four sequential levels differ from one another (p < 0.001). Applying then the within-subjects contrasts technique enables us to compare the possible couples of phase 2 HGF serum levels. There was a strong, statistically significant difference (p < 0.001) among HGF_pre basal concentrations and any of the three HGF concentrations during nadroparin therapy. The decrease between HGF_post1 and HGF_post2 was moderate , while between HGF_post2 and HGF_post3 was about 30% .We compared the same previous phase 2 HGF concentrations by stratifying them by the nature of oncological disease in the 17 patients, 10 malignant (ECOC) and 7 benign  Table . The onlIn three of our phase 2 patients, we measured basal and Cmax HGF concentration at all the observational points , and we found that at all points the basal and the Cmax concentrations differed significantly . Moreover, one of our phase 2 patients was previously under treatment with nadroparin 9500 UI anti-Xa for 11 months because of an episode of deep venous thromboembolism (VTE). In this patient, we observed an HGF concentration increase from 1.15 ng/ml before to 3.84 ng/ml after heparin administration (data not shown)., a concentration as low as 25 ng/ml was effective, but lower concentrations were never tested. Moreover, in a preclinical model, Bardella et al. (2007) demonstrated the strong effectiveness in vivo of a local concentration of 250 ng/ml, extremely higher than that obtained in our patients. We therefore tested the effectiveness of lower decreasing concentrations of recombinant HGF and found that a level as low as 1.25 ng/ml of HGF was able to sensitise ovarian cancer cells to cisplatin  and that these alterations also last after one month of daily treatment with LMWH. Therefore, treatment with calcic nadroparin might be suitable and effective when a clinical and therapeutic use of HGF is anticipated.We studied gynaecological patients with the intent of exploiting the ability of HGF to sensitise ovarian cancer cells to chemotherapeutic effects. Pre-clinical models showed that HGF enhances the apoptotic effect of low doses of PTX and CDDP on ovarian cancer cells -15. Pre-The findings reported here are important also when approaching tumour therapy from the perspective of inhibiting HGF and MET, HGF is a Janus-faced molecule, which sensitizes ovarian cancer cells to chemotherapeutics, but is alone able to trigger cancer cell growth and invasiveness. Clinical trials are ongoing, also in ECOC patients, with antibodies and small molecule inhibitor targeting HGF or MET. HGF serum increase after heparin treatment should be taken into account also in this respect.It has been definitively established that only cancer cells with specific genetic alterations, namely either met gene amplification or HGF autocrine loop maintain dependence on the met gene activation for transformation, i.e. remain met addicted. Therefore, only these cancer cells could respond to met inhibition by small molecule biochemical inhibitors or antibodies. In most instances, HGF and MET axis elicits a number of signalling pathways leading to either cell survival and invasion or cell death or differentiation.In this study, we tested calcic nadroparin because it is commonly used in our hospital for VTE prophylaxis. Surprisingly, we found that nadroparin causes an elevation in HGF concentration that is higher than most obtained with any LMWH reported in the literature. Salbach PB. et al. , for insThe way by which heparin increases HGF serum concentration has yet to be completely clarified. A hypothesis is that heparin could act by removing HGF from its binding to cell surface and matrix proteoglycans . In thisIn conclusion, our study opens the possibility of using LMWH to raise HGF serum concentration and take advantage of its biological activities. In particular, we hypothesise that nadroparin could be used as a chemo-potentiating agent in ECOC through its action on HGF serum concentration.The authors declare that there are no conflicts of interest.AS. principal investigator: contributions to conception and design, acquisition of data, analysis and interpretation of data, drafting the manuscript. LF conceived of the study, participated in its design and interpretation of data. RP participated in the design of the study and performed the statistical analysis, Pharmacokinetic Analysis, drafting the manuscript. AF contributions to conception and design of the study. CM contributions to conception and design, supervision of the research group. CM cell lines, apoptosis assay, acquisition of data. AL: ELISA determinations, acquisition of data. MFD participated in its design and coordination and helped to draft the manuscript, revising it critically for important intellectual content PZ. Acquisition of funding, participated in its design, supervision of the research group. All authors read and approved the final manuscript.Fondi per la Ricerca Finanziata dall'Università (ex 60%) 2007.


The present study investigated the effects of oral administration of annatto carotenoids (bixin (BIX) and norbixin (NBIX)) on glucose levels, lipid profiles, and oxidative stress parameters in streptozotocin (STZ)-induced diabetic rats. Animals were treated for 30 days in the following groups: nondiabetic control, diabetic vehicle, diabetic 10 mg/kg BIX, diabetic 100 mg/kg BIX, diabetic 10 mg/kg NBIX, diabetic 100 mg/kg NBIX, diabetic metformin, and diabetic insulin. Blood glucose, LDL cholesterol, and triglyceride levels were reduced in the diabetic rats treated with BIX. BIX treatment prevented protein oxidation and nitric oxide production and restored superoxide dismutase activity. NBIX treatment did not change most parameters assessed, and at the highest dose, it increased LDL cholesterol and triglycerides levels and showed prooxidant action (increased protein oxidation and nitric oxide levels). These findings suggested that BIX could have an antihyperglycemic effect, improve lipid profiles, and protect against damage induced by oxidative stress in the diabetic state. Because NBIX is a water-soluble analog of BIX, we propose that lipophilicity is crucial for the protective effect of annatto carotenoids against streptozotocin-induced diabetes. It affects approximately 6% of the population worldwide or 371 million individuals ; impairmβ cells . This diβ cells .In addition, plasma triglycerides, low-density lipoprotein (LDL), and total cholesterol levels are usually elevated in diabetes, whereas the levels of high-density lipoproteins (HDL) are reduced . The dysHyperglycemia has been hypothesized to contribute to oxidative stress either through the direct generation of ROS or by altering the redox balance . This isBixa orellana L., a native shrub from tropical America, are a rich source of antioxidant carotenoid pigments that are largely used by the food coloring industry. This pigment, which is known as annatto (E160b), contains approximately 80% bixin . The orthophosphoric acid, hydrochloric acid, potassium iodide, acetic acid, and sodium hydroxide were purchased from Vetec (Brazil). Metformin (99%) was from Valdequimica  and the NPH insulin (100 IU/mL) used was commercially purchased . All other reagents used in the experiments were of analytical grade and of the highest purity.STZ, DL-dithiothreitol (DTT), glutathione, 5,5′-dithiobis(2)-nitrobenzoic acid (DTNB), sodium nitrate, vanadium (III) chloride, sulphanilamide, N-(1-naphthyl)ethylenediamine, glutathione reductase, epinephrine hydrochloride, BIX oily solution (10%) and NBIX aqueous solution (10%) were donated by Christian Hansen (Brazil). BIX and NBIX solutions at 5 and 50 mg/mL were prepared by dilution in distilled water containing 10% Tween 80 and 0.5% ethanol. The vehicle aqueous solution contained 10% Tween 80 and 0.5% ethanol. Metformin was dissolved in water at 50 mg/mL. The solutions were prepared three times a week and stored in amber bottle at 2 to 8°C until use.Adult male Wistar rats  from the Central Animal House of the Federal University of Santa Maria were housed under controlled temperature (23 ± 1°C) and humidity (56%) on a 12-hour light-dark cycle with free access to food  and water. Before the beginning of the experiments, animals were adapted in cages for 20 days. All the experimental procedures were carried out in accordance with international guidelines for care and use of laboratory animals  and were approved by the Committee on Care and Use of Experimental Animal Resources of the Federal University of Santa Maria (protocol no.: 089/2011). Diabetes was induced by a single i.p. injection of STZ  dissolved in sodium-citrate buffer  . The conThe animals were randomly divided into eight groups (6 rats per group): nondiabetic control + vehicle, diabetic + vehicle, diabetic + 10 mg/kg BIX, diabetic + 100 mg/kg BIX, diabetic + 10 mg/kg NBIX, diabetic + 100 mg/kg NBIX, diabetic + 100 mg/kg metformin, and diabetic + insulin (4 IU). Vehicle, BIX, NBIX, and metformin were given by gavage (up to 2 mL/kg b.w.) as a daily administration for 30 days. The insulin dose was fractionated (2 IU doses) and administered intradermically twice a day for 30 days. BIX and NBIX doses were chosen based on a previous study . During After the 30 days of treatment, animals were fasted overnight and anesthetized with xylazine (10 mg/kg body weight) and ketamine (75 mg/kg body weight) and blood samples were collected by cardiac puncture into heparinized tubes to measure the activity of antioxidant enzymes. Another blood sample was collected without anticoagulant to obtain serum for the biochemical analyses and to assess the products of advanced protein oxidation (AOPP) and nitric oxide (NOx) levels.The serum concentrations of glucose, total cholesterol, triglycerides, and HDL were determined using colorimetric kits . The LDL cholesterol level was calculated according to Friedewald et al. . FructosSerum NOx levels were assessed as nitrite/nitrate content measured on Cobas MIRA as previously described . The oxiP < 0.05.The results were expressed as the mean ± SEM. Data were analyzed for statistical significance by one-way ANOVA followed by Duncan's test using the Statistica 7.0 and differences were considered significant at P < 0.05; onset levels in P < 0.05; end levels in P < 0.05; end levels in STZ-induced diabetes increased blood glucose levels 4-fold compared to the nondiabetic control rats , whereas NBIX had no effect . This occurred because only these two latter groups gained weight during the experimental period , whereas the other groups had no body weight change.No significant difference was observed in the body weight among groups at the onset of the treatment . HoweverP < 0.05; P < 0.05).Total and HDL cholesterol levels were similar between nondiabetic and diabetic rats Figures . HoweverP < 0.05; Figures P < 0.05). However, insulin or 10 mg/kg NBIX did not change LDL levels compared to the diabetic vehicle group, whereas 100 mg/kg NBIX increased these levels  to values similar to those of the nondiabetic control group and this effect was similar to that observed after insulin or metformin treatment (P < 0.05).On the other hand, the LDL cholesterol and triglycerides levels were significantly increased in the diabetic vehicle group compared to nondiabetic control rats (reatment . NBIX atP < 0.05). Treatment with BIX up to 100 mg/kg decreased the AOPP levels of diabetic rats, similar to that observed with metformin (P < 0.05). The diabetic rats treated with insulin also had no increase in AOPP levels compared to the nondiabetic control group, albeit its AOPP level was not different from the diabetic vehicle group .The oxidation of proteins, as assessed by AOPP, was increased in diabetic vehicle rats compared to the nondiabetic control group , but this decrease was recovered in all diabetic treated rats , but this change was not observed in the diabetic rats treated with BIX (10 or 100 mg/kg), NBIX (10 mg/kg), or metformin  for 30 days reduced blood glucose as well as fructosamine levels of diabetic rats. The antihyperglycemic effect of BIX seems to have occurred soon after the start of the treatment, because the decreased level of fructosamine indicates that protein glycation was reduced in the last three weeks . This ancultures . In factcultures , 34. Thecultures .α or γ receptors in this diabetes model. In fact, the cell culture studies on the activation of PPAR receptors by annatto carotenoids showed a lower potency for NBIX compared to BIX [In contrast to BIX, the treatment with NBIX did not reduce fasting blood glucose levels. The difference between these structurally similar annatto carotenoids could be related to their different polarity. BIX has a lipophilic character, whereas NBIX has a hydrophilic character. Our results suggest that NBIX had no effect in the PPAR-d to BIX , 20.α receptor, which increases the expression of genes involved in fatty acid oxidation, fatty acid uptake, and energy consumption in the liver as recently demonstrated in obese KK-Ay mice [α receptor was only preliminarily investigated in cell culture assays, where NBIX had a lower potency than BIX to activate the PPAR-α in a GAL4/PPAR-α chimera system [α receptor in the STZ model of diabetes in rats, which could be related to its lower hydrophobicity compared to BIX [In addition to the antihyperglycemic effect, BIX (10 and 100 mg/kg) completely prevented the increase in serum triglycerides and LDL cholesterol levels that occurred in diabetic rats. Moreover, the highest dose of BIX also increased the HDL levels of diabetic rats. This effect is similar to that recently demonstrated for an aqueous extract of annatto in a model of hypercholesterolemia in rats . Interes-Ay mice . This re-Ay mice  and is a-Ay mice , 39. Thea system . Thus, od to BIX .β cells and subsequent development of insulin-dependent diabetes mellitus. Moreover, several studies have shown that a single dose of STZ increases the levels of NOx, being responsible for the damage to β cells that leads to diabetes in experimental models [The exposure to high glucose levels may increase the generation of ROS through the nonenzymatic glycation of proteins and glucose auto-oxidation, which leads to oxidative stress and structural and functional tissue damage , 42. Thel models . In our AOPP is a suitable biochemical marker for measuring short-term changes in oxidative stress because it assesses the products of plasma protein oxidation, especially albumin oxidation. This marker is increased in inflammatory conditions such as diabetes, atherosclerosis, and renal failure . In the 2•−) to form peroxynitrite anion (ONOO−), which is a highly reactive oxidizing agent [2O2, because it is actively produced by superoxide dismutase but not efficiently removed by catalase. This imbalance in the removal of oxidative species was previously reported in various oxidative conditions including alloxan-induced diabetes [Antioxidant enzymes are the first line of defense against reactive species and oxidative stress . In the ng agent . Treatmediabetes  and is lin vitro, which have been attributed to the electron transfer due to the presence of many conjugated double bonds [2O2, HOCl, 1O2, and ONOO−) than for radical species (O2•− and •NO), we showed that BIX did decrease NOx levels in STZ-induced diabetes. In addition to the direct scavenging of reactive species, BIX may also enhance the antioxidant defenses, as demonstrated in the present study for superoxide dismutase, glutathione reductase, and thioredoxin reductase activities. The increased activity of these enzymes may be a result of an increased gene expression, since an annatto extract enriched in BIX was recently shown to prevent the decrease in the activity and expression of superoxide dismutase in neutrophils from alloxan-induced diabetic rats [Thus, BIX also showed antioxidant activity in the STZ-induced model of diabetes, in addition to the antihyperglycemic and antilipidemic effects. This finding is in agreement with the effectiveness of BIX and BIX-enriched annatto extracts to scavenge for both nitrogen and oxygen reactive species le bonds . Althougtic rats .BIX, at doses that show no toxic effects , was as


It is important to understand the psycho-social context of obesity to inform prevention and treatment of obesity at both the individual and public health level.2) and WC (cm) were dependent variables in separate multiple linear regression models for which predictors were entered in 4 blocks: 1. demographic factors; 2. stressful life events; 3. diet/activity; and, 4. psychological measures.Representative samples of middle-aged adults aged ≥43 years were recruited in Great Britain (GB) (n = 1182) and Portugal (n = 540) and interviewed to explore associations between body mass index (BMI), waist circumference (WC), demographic factors, physical activity, dietary habits (FFQ), life events (LES), Resilience (RS11), Mood (MS), Hopelessness (BDI) and Perceived Stress (PSS4). BMI (kg/m2) was predicted by less education, illness in a close friend or relative, frequent alcohol consumption and sedentary behaviour. Among the Portuguese, higher BMI (kg/m2) was predicted by lower resilience. Being male and less education were independent predictors of having a larger WC (cm) in both countries. Within GB, not working, illness in a close friend or relative, sedentary lifestyle and lower resilience were also independent predictors of a larger WC (cm).In the GB sample, BMI (kg/mThese data suggest that intervention to treat and/or prevent obesity should target males, particularly those who have left education early and seek to promote resilience. In GB, targeting those with high alcohol consumption and encouraging physical activity, particularly among those who have experienced illness in a close friend or relative may also be effective in reducing obesity. Whateudaimonic, which is associated with one’s sense of autonomy, purpose and personal growth or hedonic which reflects life satisfaction [Cultural differences in associations between well-being and obesity may go some way toward accounting for the conflicting findings reported in the literature. Whilst existing studies have taken place in a range of countries and cultures world-wide, there appears to be a lack of research which has compared more than one culture in the same study. There may also be sex differences in the way that wellbeing, particularly depression, interacts with obesity -18,26 ansfaction ,11. Resisfaction  and can sfaction , comprissfaction  and moodsfaction . The aimThe study was of a cross-sectional survey design conducted during autumn 2006.Sample recruitment and data collection were undertaken by Ipsos Mori (GB), an international market research company. Nationally representative samples of adults over the age of forty-three years N = 1722) were recruited in Great Britain (GB) (n = 1182) and Portugal (n = 540)  was derived from that used previously in the Zenith study of healthy ageing  and thenPhysical activity and sedentary behaviour were assessed using a scale previously employed in the Zenith study . Higher The validated 11-item version of the 25-item Resilience Scale (RSS) developed by Wagnild and Young (1993)  was usedThe validated 3-item version of the mood survey developed by Underwood and Froming (1980)  was emplThis 20-item Beck Hopelessness Scale (BHS) was originally developed by Beck and colleagues in 1974  to measuThe validated 4-item version  of the Pet al. (2000) [The 10-item Life Events Scale (LES) developed by Mooy . (2000)  was usedEthical approval for the survey was granted by the Office of Research Ethics Committee Northern Ireland (ORECNI). Fieldworkers in both countries were trained and in the employment of the market research company (IPSOS Mori). Respondents provided written (informed) consent before taking part. Omnibus survey employing a representative sample recruited in Great Britain and Portugal was undertaken during January 2006. Data were collected in each individual’s own home by interview. Cue cards were used to present the response options for each item. Anthropometric measurements were taken following completion of the questionnaire. Waist circumference was measured by means of a tape measure at the level of the umbilicus in males and at the narrowest diameter in females. BMI (kg/m²) was calculated from height measured by stadiometer and body weight by an electronic scale.2) and age (years) between country and sex. Associations between the independent variables  and the dependent variables WC (cm) and BMI (kg/m2) were examined by multiple linear regression analysis. The GB and Portugal samples were analysed separately to establish which independent variables were the best predictors of BMI (kg/m2) and WC (cm) within each country. The aim of the analysis was to establish the degree to which the psychological factors explained/contributed to the variance in BMI (kg/m2) and WC. Demographic information , life events (LES) and lifestyle factors (Dietary habits (FFQ) and physical activity (PAQ)) thus were entered first into the analysis so that each could be controlled prior to entry of the psychometric data. The predictors were entered simultaneously in 4 blocks . Continuous measures  were adjusted to assess if there were any potential interactions with sex. Variables were centred by subtracting the overall means from each individual value and then multiplying by sex to establish if there were any interactions with sex. Mahalanobis distance values indicated that there were no multivariate outliers among the independent variables  [Statistical analyses were undertaken using the SPSS . Two-way MANOVA was employed to look at differences in anthropometric and demographic characteristics, height (m), WC (cm), BMI (kg/mThe sample N = 1722) comprised in GB (n = 1182) 51% (n = 603) male and 49% (n = 579) female and in Portugal (n = 540) 47% (n = 253) male and 53% (n = 287) female. The age range for the total sample was 43–93 years (Table 722 compr2) between respondents in Portugal and GB. There were no within sex differences in BMI  = 1.32, P = 0.26)  than Portuguese males P = 0.0001) and GB females had a smaller WC (cm) than Portuguese females (P = 0.0001)  = 105.94, P = 0.0001). There were no differences in BMI (kg/m and GB f2)  = 2.36, P = 0.03). Having reported ‘illness-related’ life events (β = −0.14. P = 0.0001) explained an further 2% (i.e. 3%) of the variance in BMI (kg/m2)  = 3.36, P = 0.000). Having a high alcohol consumption  and sedentary behaviour  contributed a further 2% (i.e. 5%) of the variance in BMI (kg/m2)  = 3.91, P = 0.0001). The inclusion of the psychological variables within model 4 did not further explain the variance in BMI (kg/m2) but improved the model to 6%  = 3.07, P = 0.0001)  accounted for 1% of the variance in BMI (kg/mDemographic factors (model 1) including, sex  , fewer years in education , age*sex   and working status (unemployed)  accounted for 16% of the variance in WC  =30.692, P = 0.0001). When life events (model 2) were added to the model, ‘illness-related’ events predicted a larger WC (cm) , which together with demographic factors explained a further 2% of the variance (i.e. 18%) in WC (cm)  = 19.39, P = 0.0001). The incorporation of lifestyle factors  (model 3) indicated that greater sedentary behaviour predicted a larger WC , contributing an extra 2% of the variance (i.e. 20%) in WC  = 15.17, P = 0.0001). The addition of psychological measures (model 4) to the regression model, indicated that lower scores on resilience  predicted a larger WC (cm). Resilience, therefore, was the only significant psychological predictor variable in the final model (four) which explained 20% of the variance in WC (cm)  = 9.18, P = 0.0001). Sex had no influence upon psychological variables in determining WC (cm) (Table 2) was associated with older age  and lower resilience scores  (model 4) accounting for 1% of the variance in BMI  including, sex   and fewer years of education  accounted for 7% of the variance in WC (cm)  = 4.405, P = 0.0001). The incorporation of stressful life events (model 2) reduced the variance in WC (cm) by 1% (6%)  = 2.54, P = 0.005). Lifestyle factors (model 3) contributed an extra 1% (7%)  = 3.08, P = 0.005), indicating that greater physical activity predicted a smaller WC . Finally, the incorporation of psychological variables (model 4) weakened the model and reduced variance in WC (cm) by 1% from model 3 (6%)  = 1.63, P = 0.03)  and Portugal. The results indicated differences in which factors best explained obesity within the two countries. BMI among the British was explained by having spent less time in education, having reported illness related events, frequent alcohol consumption and lack of physical activity. The finding that BMI in GB was predicted by alcohol consumption and sedentary behaviour is in keeping with accepted science suggesting that obesity is associated with lifestyle ,8. The fWaist circumference, on the other hand, has been shown to be closely correlated with visceral fat and other factors associated with the metabolic syndrome . Being mPrevious research has suggested that certain psychological states can trigger or inhibit eating  for examPrevious studies of obesity and well-being have been biased toward the study of negative psychological states (depression and stress) to the neglect of positive traits and states that may be protective against obesity and which could afford opportunities for intervention. Negative psychological traits did not contribute to either model, bringing into question the large body of research which has indicated a link between WC ,18,25,26Our study is unusual in that it has considered in two culturally diverse representative samples of the middle aged and older normal population, both positive and negative psychological constructs and a range of demographic, social and lifestyle factors that may mediate the relationship between psychological well-being and obesity. That the GB models provided a stronger explanation for WC and BMI compared to Portugal could imply the importance of culture in understanding the interplay between psychosocial, lifestyle and psychological factors in the aetiology of obesity which may go some way toward explaining the mixed findings of previous studies. Both the GB and Portuguese models indicate the importance of targeting males and the less educated and of promoting resilience. Our data suggest that obesity prevention efforts in GB should also target care providers and the unemployed and seek to reduce alcohol consumption and increase physical activity. Meanwhile, our results add weight to the theory that research into positive psychological factors has potential to enhance understanding of obesity.The authors declare that they have no competing interests.BSK designed the study and drafted the manuscript, MD conducted statistical analyses and contributed to the manuscript, BB directed the statistical analyses, HP conducted preliminary statistical analyses; MDVA co-designed the study and MG led the research project. All authors have read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/12/424/prepub


High and low protein diets fed to pregnant adolescent sows led to intrauterine growth retardation (IUGR). To explore underlying mechanisms, sow plasma metabolite and hormone concentrations were analyzed during different pregnancy stages and correlated with litter weight (LW) at birth, sow body weight and back fat thickness. Sows were fed diets with low , adequate , and high  protein levels, made isoenergetic by adjusted carbohydrate content. At −5, 24, 66, and 108 days post coitum (dpc) fasted blood was collected. At 92 dpc, diurnal metabolic profiles were determined. Fasted serum urea and plasma glucagon were higher due to the HP diet. High density lipoprotein cholesterol (HDLC), %HDLC and cortisol were reduced in HP compared with AP sows. Lowest concentrations were observed for serum urea and protein, plasma insulin-like growth factor-I, low density lipoprotein cholesterol, and progesterone in LP compared with AP and HP sows. Fasted plasma glucose, insulin and leptin concentrations were unchanged. Diurnal metabolic profiles showed lower glucose in HP sows whereas non-esterified fatty acids (NEFA) concentrations were higher in HP compared with AP and LP sows. In HP and LP sows, urea concentrations were 300% and 60% of AP sows, respectively. Plasma total cholesterol was higher in LP than in AP and HP sows. In AP sows, LW correlated positively with insulin and insulin/glucose and negatively with glucagon/insulin at 66 dpc, whereas in HP sows LW associated positively with NEFA. In conclusion, IUGR in sows fed high protein∶low carbohydrate diet was probably due to glucose and energy deficit whereas in sows with low protein∶high carbohydrate diet it was possibly a response to a deficit of indispensable amino acids which impaired lipoprotein metabolism and favored maternal lipid disposal. The response of the conceptus to maternal food intake is mediated by the intrauterine environment of nutrients, hormones and growth factors. To explore the underlying mechanisms leading to IUGR in response to diets with imbalanced high or low protein∶carbohydrate ratios in our porcine model, it is necessary to examine the maternal metabolic response during pregnancy. Therefore, the objective of this study in adolescent sows was 1) to monitor diet-dependent changes of maternal plasma metabolite and hormone concentrations during early, mid and late stages of pregnancy, and 2) to test the hypothesis that diet-dependent changes in maternal body weight, body fat, or plasma metabolites and hormones relate to IUGR and provide clues on the underlying metabolic reasons.Procedures performed in this study were in strict accordance with the German animal protection law and approved by the relevant authorities . All surgery was performed under anesthesia (see below), and all efforts were made to minimize suffering.th parity, increasing their initial BW at first mating by approximately 60 kg to reach their full mature weight Nulliparous German Landrace sows bred at the institute's pig breeding facility were used (EXP1). Sows were estrus synchronized as recently described In EXP1, a total of 95 sows were randomly allocated to the three dietary groups. Finally, 19, 25, and 22 sows (n = 66) fed the LP, AP, and HP diets, respectively, produced a litter After overnight food withdrawal in sows of EXP1, blood samples were taken at −5, 24, 66, and 108 dpc by venipuncture. In EXP2, sows were surgically fitted with an indwelling jugular vein catheter on 84 dpc to allow diurnal blood sampling on 92 dpc. General anesthesia of sows was achieved by an i.v. administration of a combination of ketamine  and azaperone . Implantation of the jugular vein catheter was adapted to the method described by Rodriguez and Kunavongkrit g at 4°C for 20 min and the supernatant was stored at −20°C. Tubes containing F-EDTA  were used for the analysis of glucose, triacylglycerol (TG), non-esterified fatty acids (NEFA), total cholesterol (C), low density lipoprotein cholesterol (LDLC), high density lipoprotein cholesterol (HDLC), and insulin-like growth factor-I (IGF-I) concentrations. Serum monovettes  were used for the measurement of urea and serum protein levels, and tubes containing Li-heparin (14–15 kIU/l blood) were used to determine insulin, glucagon, leptin, cortisol and progesterone (P4) concentrations.Blood was immediately put on ice and centrifuged at 1500× Plasma metabolites  were analyzed by the Klinik für Rinder  using the following kits: glucose (no. 553-230) from MTL Diagnostics , NEFA (no. 434-91795) from Wako Chemicals GmbH , TG , urea (no. LT-UR 0050), and C  from Labor+Technik Lehmann . Analyses were performed automatically by spectrophotometry . Plasma LDLC (no. 969706) and HDLC (no. 650207) concentrations were measured at the Institut für Klinische Chemie und Laboratoriumsmedizin, University of Rostock, using commercial kits . The %HDLC was calculated as the proportion of plasma HDLC in % of total C concentration.3H-RIA in 10 µl duplicates as previously described Plasma insulin (no. PI-12K) and glucagon (no. GL-32K) concentrations were measured by RIA using commercially available porcine kits from Biotrend Chemikalien GmbH . The concentration of plasma leptin was analyzed by a double antibody enzyme immunoassay as described by Sauerwein et al. The glucagon∶insulin ratio was calculated as the glucagon concentration in nmol/l divided by the insulin concentration in nmol/l. The insulin∶glucose ratio was calculated as the insulin concentration in pmol/l divided by the glucose concentration in mmol/l.P<0.05. Results are reported as least square means ± SE.In EXP1, one replicate had only AP and HP sows and was excluded from the analysis. Thus, plasma data of 16, 17, and 15 sows in the LP, AP, and HP group, respectively, were evaluated, if not given otherwise. In EXP2, plasma metabolite concentrations of 27 sows  were analyzed. Data was evaluated using SAS/STAT 9.2 . Repeated measure ANOVA (PROC MIXED) was used to evaluate the effects of diet on sow plasma metabolite and hormone concentrations. The model included the fixed factors diet, replicate, the repeated factor time during pregnancy, and the interactions diet×replicate, and diet×time. Post hoc comparisons between different diet groups were made using the Tukey-Kramer test. The CORR procedure was used to calculate Pearson correlation coefficients within diets and pregnancy days between plasma metabolite and hormone concentrations and sow BW, sow BF as well as pregnancy outcome . The significance level was set at andP<0.01). In LP sows, LDLC concentrations were lowest compared with AP (P<0.01) and HP sows (P<0.01) (P<0.001) and decreased in LP sows (P<0.01). In addition, serum protein concentration was lower in LP compared with AP (P<0.01) and HP sows (P<0.05) (P<0.01). The diet effect within replicates differed in magnitude but not in direction of the effect. With the exception of plasma glucose and NEFA concentrations, diet×time interactions were or tended to be significant for all other variables (andP<0.05). In addition, plasma %HDLC was lowest in HP sows at 24 and 66 dpc, and higher before than during pregnancy irrespective of the diet (P<0.05) (P<0.05). Serum protein concentration decreased from mid to late pregnancy in LP and AP sows, and was lower in LP compared with AP and HP sows in late pregnancy (P<0.001)  whereas IGF-I concentrations were lower in LP than in AP sows at 24 and 66 dpc (P<0.05). Concentrations of P4 were higher in HP than in LP (P<0.05) with intermediate values in AP sows. Cortisol levels were lower in HP compared with AP sows (P<0.05). In late pregnancy, cortisol concentrations were increased only in LP and AP sows compared with early and mid pregnancy  as compared with AP sows (P<0.001), whereas in LP sows urea levels were approximately at 60% of the control values (P<0.001) (P≤0.1) (P<0.01) and LP sows (P<0.001) (P<0.01) and HP sows (P<0.001) , mid (66 dpc), or late (108 dpc) pregnancy are shown in In regard to BW, in AP sows at 24 dpc there was a negative correlation with NEFA Table 6.In LP sows, negative correlations were detected at 24 dpc between BF and serum protein, and BF and LDLC, whereas positive correlations were observed between BF and insulin and insulin∶glucose ratio. HP sows showed a negative relationship between BF and NEFA Table 6.No correlations at all were observed between sow BW and LW. There was a significant negative relationship between LW and C at 24 dpc in LP sows Table 6.There is insufficient data available on the long-term effect of high protein diets, especially in reproductive females. We observed IUGR in offspring from sows fed a diet with high protein∶carbohydrate ratio, similar to that found in sows fed a low protein diet throughout pregnancy Our results indicate that IUGR in the HP group was associated with a low energy status as suggested by 1.8 times the diurnal plasma NEFA concentrations of control sows. This corresponds to a NEFA increase of a similar magnitude as observed after 24 h fasting in pigs The higher basal plasma glucagon and lower diurnal glucose levels suggest a maternal glucose deficit in HP sows. This interpretation is supported by the low body fat content and compromised body mass gain in this pig model Adaptive responses in nitrogen metabolism such as decreased plasma urea concentration were apparent early in pregnancy of AP and LP sows. This effect is also known in humans and reflects a reduced urea synthesis associated with protein accretion by the mother Diet-dependent differences in postabsorptive plasma concentrations occurred in relation to lipoprotein and C metabolism. Lowest HDLC values were observed in HP sows whereas LDLC concentrations were lowest in LP sows as compared with control sows. This suggests divergent alterations in the lipoprotein metabolism of LP and HP sows. Since the experimental diets were comparably low in fat and free of C In fetal development, C is important as an essential structural component of cell membranes and thus fetal growth. Maternal plasma lipoprotein C could play a nutritive role in embryonic and/or fetal development. It has been shown that a low maternal HDLC concentration was related to a 15% reduced fetal mass, and sterol homeostasis in fetal tissues was affected by maternal plasma C concentration Plasma P4 is produced by the corpus luteum graviditatis and is responsible for implantation, early embryo development and maintenance of pregnancy. In agreement with earlier findings in sows, P4 increased in the maternal plasma during the first four weeks of pregnancy and decreased slowly thereafter In spite of the difference in body fatness among the three groups of sows The HP diet caused lower total cortisol concentrations in plasma compared with control sows, an effect which was also observed in fasting rats after feeding a high protein∶low carbohydrate diet over a period of 6 months The IGF system is considered as one of the most important regulators of fetal growth but the extent to which IGFs influence the mother and/or placenta are dependent on the species and maternal factors, including nutrition Overfeeding during adolescent pregnancy in ewes has been shown to be associated with an adverse outcome for the offspring


Plasmodium vivax, little is known about its transmission dynamics. This study explored the population structure and spatio-temporal dynamics of P. vivax recurrent infections after radical cure in a two-year cohort study carried out in a rural community of the Peruvian Amazon.Despite the large burden of P. vivax participants recruited in San Carlos community (Peru) between April and December 2008 were treated radically with chloroquine and primaquine and followed up monthly for two years with systematic blood sampling. All samples were screened for malaria parasites and subsequently all P. vivax infections genotyped using 15 microsatellites. Parasite population structure and dynamics were determined by computing different genetic indices and using spatio-temporal statistics.A total of 37 P. vivax infections, most of them sub-patent and asymptomatic. The parasite population displayed limited genetic diversity (He = 0.49) and clonal structure, with most infections (84%) being monoclonal. Spatio-temporal clusters of specific haplotypes were found throughout the study and persistence of highly frequent haplotypes were observed over several months within the same participants/households.After radical cure, 76% of the study participants experienced one or more recurrent P. vivax recurrences were commonly observed after radical treatment, and characterized by asymptomatic, sub-patent and clustered infections . Moreover low genetic diversity as well as parasite inbreeding are likely to define a clonal parasite population which has important implications on the malaria epidemiology of the study area.In San Carlos community,  Plasmodium vivax, the most widely distributed malaria species, threatens almost 40% of the worldwide population [P. vivax population structure and dynamics are major obstacles to achieve effective malaria control, particularly in low endemic areas [P. vivax population genetic studies to understand how parasites are circulating in the human population, as well as related topics such as the spread of drug resistance and the development of clinical immunity [P. vivax populations in the Peruvian Amazon are extremely heterogeneous, with different degrees of diversity and polyclonality [P. vivax cohort conducted within 21 rural communities, and which results will be published later. In this paper, one of these study villages has been selected in order to analyse, at community and household level, the population structure and the spatial-temporal dynamics of P. vivax infections after radical treatment of the primary infection.pulation . Vivax mpulation  and, dueic areas . In the ic areas . Both chic areas . Moreoveic areas -8. Thereimmunity ,10. Simiimmunity -13, P. vlonality ,15. ThisP. vivax, and Anopheles darlingi is the main vector [The study was conducted in San Carlos (latitude 3°57′30.99″S longitude 73°20′45.76″W), a relatively small and isolated village in the Peruvian Amazon, situated at 10 km from the main road (Iquitos-Nauta road) and 21 km southeast of Iquitos city (six hours by boat via the Itaya river)   followed by active case detection of all fever cases (April to December 2008), following the study protocol of the larger cohort study. All microscopically confirmed P. vivax-infected participants were treated (directly observed treatment) with CQ 25 mg/kg/day (three days) and PQ 0.50 mg/kg/day (seven days), following the national guidelines for radical cure [P. vivax recurrent infections. Study subjects were visited at home weekly for the first month after treatment, and then monthly for the next 23 months. If a participant missed one scheduled sampling, the health care worker came back to the community two more times to look for the participant and/or scheduled the most feasible time to visit. At each visit, the medical staff asked study participants about any symptoms they may have had since the previous visit. A blood sample for microscopy (thick and thin film) and a blood spot on filter paper (BSFP)  for later molecular analysis were systematically collected. Between scheduled visits, participants were encouraged to consult the study team whenever they felt ill, to check for fever and malaria infection (blood smear and BSFP). If malaria parasites were identified by microscopy in the field after recruitment, participants were treated with CQ for three days under direct supervision, and followed up until parasite clearance (confirmed by microscopy). Those participants with 2 or more episodes of malaria after the recruitment were given a complete 7-day PQ treatment before the 2 year follow up. This study was approved by the Ethical Boards of Universidad Peruana Cayetano Heredia, Peru , the Institute of Tropical Medicine Antwerp and the University Hospital of Antwerp (Belgium).cal cure . For thiPlasmodium species-specific PCR (ssPCR) to confirm the presence and density of P. vivax parasites, and this was repeated for all blood samples taken during participants’ monthly follow-up (scheduled and unscheduled visits). Parasite density was computed after counting the number of asexual parasites for 200 white blood cells (WBC) in the thick smear and assuming a concentration of 8,000 WBCs/μl. A slide was declared negative if no malaria parasite was found after examining 100 fields. Quality control was done by a senior technician on all positive and 10% of negative slides [All day 0 (D0) blood samples were analysed by microscopy (thin and thick film) and e slides .Plasmodium and mammal sequences, and obtained amplicons were used for a second PCR using Plasmodium-species specific primers.Parasite DNA was extracted from the BSFP using the saponin lysis Chelex 100 method , and eitPositive vivax malaria samples, either by microscopy and/or species specific PCR, were selected for genotyping using 15 neutral, or nearly neutral, microsatellite markers (MS) as described elsewhere ,23. ThisP. vivax recurrence was defined as any symptomatic  or asymptomatic P. vivax infection identified by microscopy and/or PCR occurring between seven days and 24 months after the start of radical cure treatment. Recurrences were defined as “patent” if identified by microscopy, and “sub-patent” if detected only by ssPCR. All sub-patent infections were assumed to have an average parasite density of 10/μl [t-test.Data were double entered and cleaned in Excel  and then analysed with SPSS v.16 . A An infection was defined as polyclonal  if at least one locus presented more than one allele . The perHe)) were computed for each population using FSTAT v.2.9.3 [He represents the probability of finding a different allele for a given locus in any pair of haploids randomly drawn from the same population. Genepop v4.0 software was used to assess the population differentiation through the analysis of the allele distribution [The total number of alleles per each locus, the allelic richness , the number of alleles detected in a sample by any locus, and the genetic diversity of each locus  was computed with LIAN 3.5 software [IAs was assessed among monoclonal infections, respectively for D0, monthly recurrences and all samples together. In addition, pair-wise linkage disequilibrium (pair-wise LD) was assessed between every pair of markers using G-statistics with FSTAT software [psex) in two or more samples was computed using GenClone ver. 2.0 [The standardized index of association  were used to identify clonal parasite groups. eBURST software v.3  was used 11 loci . The hapver time . In addiver time . The rel.4.6.1.0 .p-values were obtained through Monte Carlo simulations [Using the geo-coding of the participants at household level and the SaTScan software v9.1.1 , space-tulations .P. vivax mono-infection and recruited into the study. Males (n = 19) and females (n = 18) were equally represented, and the median age was 15 years (IQR 3–48) with 40% of study participants aged < nine years. At D0 the mean parasite density was 2,031/μl . Though all D0 slides were double-read by a senior technician, the ssPCR remained negative (ssPCR repeated once after negative amplification) for two participants whose parasite density by microscopy was 48/μl and 823/μl, respectively. During the first 28 days of follow-up, there was no P. vivax recurrence observed by microscopy or by ssPCR , the prevalence of vivax malaria in San Carlos was estimated at 10.2% by standard microscopy. Between April and December 2008, a total of 37 individuals (in 20 households) were identified with a P. vivax recurrent infections were detected by microscopy with a mean parasite density of 88/μl (95% CI: 56–139) which was significantly lower than that at D0 (p < 0.001). The ssPCR missed two recurrent infections despite a parasite density by microscopy of 36 and 4,352/μl, respectively. By the end of the two-year follow-up, 76% (28/37) of the study subjects had experienced at least one recurrent P. vivax infection, while only two Plasmodium falciparum infections were identified. More than half of the recurrent infections  were sub-patent and asymptomatic.At day 28, all 37 participants were found negative both by microscopy and ssPCR. During the following 23-month period, a total of 142 P. vivax infections (35 D0 and 139 recurrences) were genotyped by MS PCR  was 0.49 (range 0.10-0.67) (Table He = 0.65) discriminating 58.6% of all polyclonal infections. The average total number of alleles per locus was 4.6 (range 2–7), the average allelic richness was 4.17 (range 1.99-6.28) and the average number of alleles detected in a sample by any locus remained constant between all loci (1.1-1.2). Average genetic indexes did not vary between D0 and recurrences, however looking at specific periods of the study, differences were found, as shown later.A total of 174 R Figure , among wIAs = 0.51, p < 0.0001) indicating a clonal population. To check for bias due to unequal distribution of the haplotypes or due to sampling, the LD was also assessed for unique haplotypes only, and it remained significant . Inbreeding, rather than physical linkage between markers established in the same contig , was identified by the linkage between multiple loci obtained by pair-wise LD analysis .The analysis of the monoclonal samples n = 136) showed a highly significant overall LD  did not vary substantially between baseline (D0) and first year monthly follow-up samples, 0.44 and 0.40 (p = 0.26), respectively. Nevertheless, He was significantly higher during the second year of follow-up (He = 0.51) compared to both baseline and first year (p < 0.0001). In addition, compared to the first year, multilocus LD (IAs) during the second year decreased from 0.6 to 0.4. The frequency of the most common haplotype at D0 and during the first year, i e, H11 (=haplogroup D), decreased substantially during the second year , while in the same period new infrequent haplotypes  and other haplotypes belonging to the haplogroup A were observed  were identified during the study period Figure . ClusterMore than two thirds of the participants  had infections with one to two different haplotypes during their follow-up period, while the remaining had infections with three to five haplotypes (up to three different haplogroups or singletons). The most frequently detected haplotype, H11, dispersed within 80% (16/20) of the participants’ households and occurred in 60.7% (17/28) of the participants in at least one infection (up to eight infections with H11 in two participants).One or more sub-patent recurrent infections that cleared without treatment were observed in 50% 14/28) study subjects  found in San Carlos indicates that inbreeding was the most likely reproductive event in this area. This was also the case in other reports from endemic areas of the Amazon Basin [In San Carlos, a relatively isolated village in the Peruvian Amazon, the majority (76%) of the 37 study participants experienced ountries ,36-39. TColombia  but diffColombia  and otheColombia ,12. ThisColombia . Though Colombia ,42,43, ton Basin ,13,43. IP. vivax[Clustering of malaria transmission at household level has been previously reported in the Peruvian Amazon ,14. In tP. vivax,45, can P. vivax.P. vivax microsatellite diversity is produced within the human host during the mitotic replication [The clustering of haplotypes may also influence the parasite diversity and the interpretation of recurrent parasitaemia. Some participants carried the same haplotype for several consecutive months, and later presented a new but closely related haplotype . Two other peaks (K = 6 and K = 9) are described with a lower likelihood. K = 6 corresponded to the six haplogroups defined by eBURST when the criteria of relatedness was increased to 14 loci instead of 11. Panel B illustrates the bar plot at K = 4 with 136 samples being represented by a single vertical line divided into colours assigned to the population of origin. Each colour represents one population , and the length of the coloured segment shows the estimated proportion of membership of that sample to each population.lsewhere , the uppClick here for fileCharacteristics of each malaria infection suffered by the 37 participants. X = Initial infection in this study (D0). Green shading indicates a recurrent P. vivax infection. P = patent infection. On the upper left corner of the cells is defined the haplotypes (H1…H20) or one of the symbols explained below. i = incomplete allele data, m = mixed infections (not possible to differentiate the haplotypes). ind = haplotype within a mixed infection that cannot be determined. n = P. vivax sample that did not amplified by PCR with any of the microsatellites. † Patent infection detected by microscopy but no by ssPCR (MS genotyping was not done). ‡ Pv positive by microscopy on D0 but filter paper D0 was not available. On day 1 was negative by ssPCR (MS genotyping was not performed).Click here for file


Gammaproteobacteria ; however, clones associated with Betaproteobacteria (10.9%), Alphaproteobacteria (9%), and Firmicutes (1.9%) were also identified. Pseudomonadaceae was the most common family affiliated with these clone sequences. The sequences were further analyzed by MOTHUR, yielding 81 operational taxonomic units (OTUs) grouped at 97% stringency. Richness estimators also calculated by MOTHUR indicated that oil samples with high-water content were the most diverse. Comparison of bacterial communities present in these four samples using LIBSHUFF and Principal Component Analysis (PCA) indicated that the water content significantly influenced the community structure only of crude oil obtained from PA. Differences between PA and PB libraries were observed, suggesting the importance of the oil field as a driver of community composition in this habitat.Crude oil samples with high- and low-water content from two offshore platforms (PA and PB) in Campos Basin, Brazil, were assessed for bacterial communities by 16S rRNA gene-based clone libraries. RDP Classifier was used to analyze a total of 156 clones within four libraries obtained from two platforms. The clone sequences were mainly affiliated with  Biodegraded oils resulting from the action of microorganisms that destroy hydrocarbons and other oil components have been a problem for the petroleum industry. Biodegradation is responsible for the increase in viscosity and acidity of the oil and the reduction of its API (American Petroleum Institute) grade . Differe During the aging of oil fields, industries make use of secondary oil recovery (SOR), which consists of water injection inside the reservoir to maintain the formation pressure, resulting in an increase of water content in crude oil (oil : water ratio of the production fluids). SOR and high-water content in the petroleum reservoir may reduce internal temperature and allow the biodegradation of crude oil by autochtone or allochtone bacterial populations . To cont Despite the recent use of molecular techniques for a broader survey of microbial communities in oil fields, our knowledge of the nature and diversity of bacteria present in these ecosystems is still scarce, especially in waterflooded oil reservoirs. Therefore, in this study, the bacterial communities from crude oil samples containing high- and low-water content from two platforms in Campos Basin were analyzed using the 16S rRNA gene sequencing approach in order to determine whether the different water content could influence the bacterial communities from crude oils and, consequently, pose a risk of decreasing oil quality.2. The oil densities from these fields range from 20 to 26 API degrees. The temperatures of the Caratinga and Barracuda fields at 2,800 m below sea level are 78 and 79.5°C, respectively. Seawater was injected into these platforms in 2005 for SOR. Two wellheads were selected within each platform, one with high-water content and another with low-water content. The wellheads were denoted as PAH, PAL, PBH, or PBL, corresponding to platform PA or PB and high- (H) or low- (L) water content . The crude oil samples were collected in sterile 1 L flasks directly from each platform wellhead after enough crude oil was drained off to clean the wellheads. Samples were stored at 4°C until their arrival at the laboratory.The Caratinga and Barracuda oil fields are located in the south-central portion of the Campos Basin approximately 90 km offshore of Rio de Janeiro State, Brazil, at water depths ranging between 600 and 1200 m. The Caratinga and Barracuda oil fields are denoted as platform A (PA) and platform B (PB), respectively. These two oil fields cover a total area of approximately 234 kmCrude oil samples (10 mL) were mixed with an equal volume of Winogradsky buffer  and incuEscherichia coli small subunit rRNA genes. The 50 μL PCR reaction contained 50 mM KCl, 2.5 mM MgCl2, 2 mM dNTPs, 0.2 μM of each primer (U968: 5′AACGCGAAGAACCTTAC3′ and L1401: 5′GCGTGTGTACAAGACCC3′), 2.5 U of Taq DNA polymerase , and 1 μL (20–50 ng) of the DNA extract. The amplification conditions included a denaturing step at 94°C for 2 min followed by 35 cycles at 94°C for 1 min, an annealing step at 48°C for 1 min and 30 s, and an extension step at 72°C for 1 min and 30 s; the final extension step was performed at 72°C for 10 min. Negative controls (without DNA) were also included in all sets of PCR reactions. PCR products were then purified using the Wizard PCR Clean-Up System (Promega) and eluted with 50 μL of distilled water. The presence of PCR products was confirmed by 1.4% agarose gel electrophoresis.16S rRNA gene sequences were amplified by polymerase chain reaction (PCR) using the primers and the PCR conditions described previously . PrimersEscherichia coli JM109 competent cells, and clones containing inserts were sequenced. All sequencing reactions were performed by Macrogen Inc. (South Korea). The sequence data set was screened for potential chimeric structures by using Bellerophon [http://greengenes.lbl.gov/cgi-bin/nph-bel3_interface.cgi)). A total of 156 valid 16S rRNA gene sequences were analyzed for taxonomic affiliation by the RDP Classifier [The partial 433-bp 16S rRNA gene sequences obtained by PCR were cloned using the pGEM T-easy vector according to instructions from the manufacturer (Promega). The resulting ligation mixtures were transformed into lerophon  (availab 16S rRNA gene sequences were then clustered as OTUs at an overlap identity cut-off of 97% by MOTHUR software . Richneshttp://www.mothur.org/wiki/Libshuff) [Based on the sequence alignment mentioned above, a distance matrix was constructed using DNAdist from PHYLIP (version 3.6) , and paiibshuff) . AdditioAfter recovering the microbial fraction, DNA was successfully extracted from the crude oil samples. However, a low amplicon yield was obtained with the bacterial 16S rRNA gene primers used, probably due to the inefficiency of DNA extraction from oily samples. As expected, no amplicons were obtained from the blank tube. The PCR products were cloned, and a total of 156 valid 16S rRNA gene sequences were analyzed for taxonomic affiliation by the RDP Classifier. Gammaproteobacteria, and most were associated with the family Pseudomonadaceae (105 clones). The remaining clones (17) were affiliated with Moraxellaceae, Enterobacteriaceae, Alteromonadaceae, and Xanthomonadaceae. Also, 17 (10.9%) of the total clones were Betaproteobacteria, and most were associated with the families Comamonadaceae (3 clones from samples PAH and PBL), Burkholderiaceae (3 clones from PAL and PBH), and Oxalobacteraceae . A few clones (14) from the PBH sample, corresponding to 9% of the total amount of clones analyzed, were affiliated with SAR11 clade, a lineage of bacteria from the Alphaproteobacteria class, which is common in the ocean [Bacillaceae  and Veillonellaceae (one clone from sample PBH) were present, representing the Firmicutes.Among the clones, 122 (78.2%) were Gammaproteobacteria.All 16S rRNA gene sequences were then clustered as OTUs, and using the 81 resulting OTUs, a phylogenetic tree was constructed with the closely related sequences that were recovered from the GenBank database . The phyP = 0.001). PAL and PAH libraries also were statistically different. However, LIBSHUFF did not show statistical differences between PB libraries (PBL and PBH) nor between samples with low water content (PAL and PBL). This latter finding was similar to the PCA result  were statistically different  and Barracuda (PB) fields. In addition, water injection might have stimulated bacterial growth by supplying nutrients and decreasing downhole temperature, as high-water content samples  showed higher richness index values . Most lie oceans . TherefoPseudomonadaceae was observed in the four 16S rRNA gene libraries. The presence of this group may be a potential risk for petroleum biodegradation inside the reservoir, because members of this family have been isolated and also detected by molecular techniques in deep ocean crust and in high temperature oil reservoirs [Pseudomonas has been shown to degrade alkanes and other polyaromatic hydrocarbons as well as to emulsify and degrade resins from Arabian light crude oil [P. putida is a metabolically versatile bacterium that is able to degrade a wide variety of xenobiotic compounds. Also, Chayabutra and Ju [P. aeruginosa, degrades n-hexadecane under anaerobic denitrifying conditions.An abundant group belonging to the family servoirs , 25. Morrude oil . Nelson rude oil  demonstra and Ju  showed tPseudomonadaceae, clone sequences affiliated with Moraxellaceae, Enterobacteriaceae, Alteromonadaceae, and Xanthomonadaceae families were also found in the oil samples studied. Some members of these families have been shown to be involved in hydrocarbon degradation [Betaproteobacteria-related clones belonging to the Burkholderiales order were observed in crude oil samples from Barracuda and Caratinga fields. Some genera of this group (Burkholderia and Comamonas) have been previously found to grow under anaerobic conditions in petroleum reservoirs or in contaminated soils [Marinobacter, Burkholderia, and Pseudomonas in water used in SOR.In addition to radation –32. Betaed soils , 33. Theed soils  have alsBacillus strains have been previously isolated from oil reservoirs in Brazil [Pelosinus-related clone, was observed in a petroleum environment. Bacillus and Pelosinus species may also survive in a petroleum environment due to spore formation or use of nitrate or iron for respiration.Few clones related to the Firmicutes phylum were detected in samples PAH, PBH, and PBL. Within this phylum, n Brazil , 34, whePseudomonadaceae, which was observed in all crude oil samples. Moreover, few clones were related to genera that have not been described before in this environment. However, we are aware that a limited number of clones were evaluated in this study. Bacterial isolation is still necessary to determine the industrial and ecological significance of these different bacteria.In conclusion, we report the bacterial composition present in crude oil samples with high- and low-water content from two Brazilian oil fields, Caratinga and Barracuda. This information on bacterial diversity in crude oil increases the current knowledge of the microbial ecology in this environment, which may help to predict the potential for biodegradation in these fields. Based on their 16S rRNA sequences, most of the clones obtained were related to different bacterial families, with a predominance of


Cancer vaccine is one of the attractive treatment modalities for patients with castration-resistant prostate cancer (CRPC). However, because of delayed immune responses, its clinical benefits, besides for overall survival (OS), are not well captured by the World Health Organization (WHO) and Response Evaluation Criteria in Solid Tumors (RECIST) criteria. Several surrogate markers for evaluation of cancer vaccine, including prostate-specific antigen doubling time (PSADT), are currently sought. The purpose of this study was to assess prospectively the PSA kinetics and immune responses, as well as the efficacy, safety, and biomarkers of personalized peptide vaccination (PPV) in progressive CRPC.One hundred patients with progressive CRPC were treated with PPV using 2–4 positive peptides from 31 candidate peptides determined by both human leukocyte antigen (HLA) class IA types and the levels of immunoglobulin G (IgG) against each peptide. The association between immune responses and PSADT as well as overall survival (OS) was studied.p < 0.0001 and p = 0.0007, respectively), which in turn showed correlation with OS (p = 0.018). Positive IgG responses and prolongation of PSADT during PPV were also significantly associated with OS (p = 0.001 and p = 0.004) by multivariate analysis.PPV was safe and well tolerated in all patients with a median survival time of 18.8 months. Peptide-specific IgG and T-cell responses strongly correlated with PSADT (PSADT could be an appropriate surrogate marker for evaluation of the clinical benefit of cancer vaccine. Further randomized trials are needed to confirm these results.UMIN000001850 Changes in serum prostate-specific antigen (PSA) can reflect the burden of disease and clinical benefit in patients with castration-resistant prostate cancer (CRPC) with cytotoxic chemotherapy or hormonal agents known to kill tumor cells; these changes can have practical utility by providing and updating prognostic information on an individual patient over time -4. As obPersonalized peptide vaccine (PPV) uses multiple peptides based on the pre-existing immunity. Under PPV treatment, each patient with human leukocyte antigen (HLA)-class IA types positive was tested for their immunological reactivity to 31 different peptides capable of inducing T-cell responses. The 31 peptides were derived from a number of tumor associated antigens: PSA, prostatic acid phosphatase (PAP), prostate-specific membrane antigen (PSMA), multidrug resistance protein and a variety of other epithelial tumor antigens. We previously demonstrated that PPV was safe and improved OS with immune responses in phase I, I/II, and II clinical trials in patients with CRPC -16. HoweEligibility required a histological diagnosis of prostate adenocarcinoma and progressive disease (PD) defined as at least two consecutive increases in PSA, new metastatic lesion on radionuclide bone scan, or progressive tumor lesions on cross-sectional imaging, despite adequate androgen ablative therapy. Patients showed positive IgG responses to at least two of the 31 different candidate peptides .All patients underwent relevant radiologic studies and bone scans every 6 months or at the progression of symptoms. PD was defined as radiographic progression evaluated by Response Evaluation Criteria in Solid Tumors (RECIST) criteria  or clini2/b, where b denotes the least square estimate of the linear regression model of the log-transformed PSA values on time. For analytical purposes, negative PSADT estimates and high positive PSADT estimates (>36 months) were censored at 36 months.To assess the PSA response for each patient, percent PSA change from baseline was calculated for each phase of the study (pre- and during vaccination). In addition, PSA doubling time (PSADT) was calculated using all serum PSA values for a specified period, and using a minimum of three PSA values by the formula logTo investigate biomarkers for OS that may allow patient selection and prediction of a response to PPV, serum amyloid A (SAA), C-reactive protein (CRP), and interleukin (IL)-6 in plasma at baseline were additionally examined by enzyme-linked immunosorbent assay (ELISA), respectively.To study the humoral responses specific to the vaccinated peptides, peptide-specific IgG levels were measured by a Luminex system , as reported previously . If the 5 x PBMCs in response to the vaccine peptides and those to the control peptide at pre- and 6th vaccination; at least 2-fold more spots at the 6th vaccination than at pre-vaccination was considered positive.Although T-cell subsets using flowcytometry was not analyzed in this study, T-cell responses specific to the vaccinated peptides were evaluated by IFN-γ ELISPOT assay using peripheral blood mononuclear cells (PBMCs), as reported previously . PeptideAll patients who received more than 6 vaccinations were considered evaluable for tumor response, and all patients entered were included in the survival analysis. Data were analyzed at the end of November, 2012 using commercially available computer software. The Student’s t-test and the chi-square test were used to compare quantitative and categorical variables, respectively. Survival was calculated from the date of first treatment until the date of any cause of death. Patients lost to follow-up were censored at the last known date of survival. The Kaplan-Meier method was used to estimate actuarial survival curves, and groups were compared using a log-rank test. Cox proportional hazards regression model was used for univariate and multivariate analyses to identify factors that had a significant impact on survival. All baseline parameters in the survival and proportional hazards regression analysis were analyzed as dichotomous variables using median or cut-off values. A two-sided significance level of 5% was considered statistically significant.Between April 2009 and August 2011, 100 patients with CRPC were enrolled in this trial at Kurume University Hospital. All 100 patients received at least one vaccination with a median of 16 vaccinations  and were included in the safety assessment and survival analysis. Three patients did not complete 6 vaccinations (1 cycle) and were excluded from the assessment of PSA response and immune responses. The reason for these failures to complete 6 vaccinations was withdrawal of consent. The median age of participants was 69 years , and the ECOG performance status was 0 in 91of the patients and 1 in the remaining 9. The median PSA and pre-vaccination PSADT at the entry to the study was 29.8 ng/ml  and 2 months , respectively. Fifty-seven patients had a Gleason score of ≥ 8 and 86 patients had metastasis. All patients had experienced progression after androgen deprivation therapy as an initial or secondary therapy. Forty patients had received docetaxel based chemotherapy with a median cycle of 6.5 as a third line treatment. Baseline patient characteristics are shown in Table The overall toxicities are shown in Table p = 0.035)   and those in the other group at 5 to 10 months (p < 0.005) during the PPV. In addition, average% PSA changes in the responder group showed a trend of PSA plateau. Average% PSA changes from baseline among three groups before and during PPV are shown in Figure Forty-eight (49%) patients exhibited some decrease in PSA from baseline, ranging from 1.9% to 99.6% . At the time of analysis with a median follow-up of 18 months , 64 deaths had occurred. Median survival time was 18.8 months  in all patients. Median survival time in chemotherapy naive patients and in patients after docetaxel chemotherapy were 21.6 months and 11.6 months, respectively.The number of selected peptides were 4 peptides in 62 patients, 3 peptides in 17 patients and 2 peptides in 21 patients at the first screening. Same peptide at the first screening were only selected in 29 of 97 (30%) patients at second screening and in 10 of 66 (15%) patients at the third screening, remaining patients received at least 1 different peptide during the study. The most frequently selected peptides were Lck486 (40 patients), CypB129 (31 patients), PAP213 (24 patients), SART2-93 (21 patients), PSA248 (20 patients), Lck488 (17 patients) and WHSC2-123 (16 patients) at the first screening. All 31 peptides were selected at any screening in the study.p < 0.0001) and CTL  responses  patients, 62 of 66 (94%) patients, 36 of 36 (100%) patients, 16 of 16 (100%) patients, and 7 of 7 (100%) patients at the 6th, 12th, 18th, 24th, and 30th vaccinations, respectively. Finally, positive IgG responses during PPV were observed in 76/97 (79%) patients. PBMCs from 97 patients were available for IFN-γ Elispot assay at the pre- and 6th vaccination. Peptide-specific T-cell responses were detectable in 42 patients (43%) at the 6th vaccination. There was no obvious correlation between IgG and CTL responses. Positive immune responses of both IgG and CTL based on baseline characteristics including age, PS, HLA typing, PSA, Gleason score, presence of metastasis and prior chemotherapy are shown in Figure % patientp < 0.0001), positive IgG response (p < 0.0001), low CRP (p = 0.012), prolongation of PSADT (p = 0.018), low PSA (p = 0.004), prior chemotherapy status (p = 0.037), positive T-cell response (p = 0.039), and presentation of ≥50% PSA decline (p = 0.046) were significantly associated with survival.Cox proportional hazards regression analysis was performed to determine factors that would predict disease death Table . Univarip less than 0.05 in the univariate analysis were included in multivariate analysis of the model. Finally, positive IgG response (p = 0.001) and prolongation of PSADT (p = 0.004) during PPV, as well as baseline good performance status (p = 0.004), low CRP levels (p = 0.006), and low PSA levels (p = 0.008), were significantly favorable factors for OS (Table The factors showing  p = 0.00 during Pp = 0.018). These results suggest that the development of late immune responses is associated with changes in PSADT.As observed in several clinical trials, immunotherapy can induce novel patterns of antitumor responses distinct from those of chemotherapy, which are consequently not captured by the WHO or RECIST criteria . On the The evaluation of T-cell immune responses to target self antigens after vaccine clinical trials presents several challenges. Antigen-specific T-cells can be evaluated by their peptide target specificity, proliferative capacity, cytokine secretion, cytolytic activity, and membrane markers of activation. At present, the best measure of antigen-specific T-cells is unknown, as is the optimal time to evaluate immune responses. In our current analysis, we evaluated both humoral responses determined by peptide-specific IgG levels using a Luminex system and antigen-specific CD8+ T-cell responses by using IFN-γ ELISPOT assays, to provide a more direct quantitative assessment after immunization. Delayed 50% PSA decline and prolongation of PSADT were observed in patients with positive IgG and T-cell respkonses, and these immune responses were associated with OS. These results suggest that further immunological analysis at multiple time points might be needed to determine whether T-cell response or the development of late immune responses is associated with clinical responses.Cancer vaccinations do not always extract good immune and/or clinical responses in vaccinated patients. This study showed that IgG responses and prolongation of PSADT during PPV, along with baseline performance status, CRP, and PSA levels, were well correlated with OS in patients with CRPC treated by PPV. These results suggest that risk stratification based on these factors could be helpful for estimating the OS in patients with CRPC treated by immunotherapy.Despite these encouraging observations, the current study must be interpreted as hypothesis-generating due to several limitations. This single-arm phase II study without a concurrent control arm did not allow estimation of the potential clinical or immune effects of this treatment. Another potential limitation of this study regarding OS is the lack of treatment data after the treatment phase of the trial. Imbalances due to chance may have occurred in treatments after progression. However, only docetaxel has been shown to affect survival in this population of patients, and only by a few months. The median survival of 18.8 months  observed in this study surpassed the survival that was observed from docetaxel-based clinical trials in a similar population by TAX-327  and South West Oncology Group 9906  [This study showed that PPV in patients with CRPC was active and well tolerated, improving survival with immune responses, delayed PSA responses, and prolongation of PSADT. Further randomized trials are needed to confirm these preliminary results.CR: Complete response; CT: Computed tomography; CRPC: Castration-resistant prostate cancer; CTL: Cytotoxic T lymphocytes; EOCG: Eastern cooperative oncology group; HLA: Human leukocyte antigen; IFN- γ: Interferon-γ; IgG: Immunoglobulin G; OS: Overall survival; PBMC: Peripheral blood mononuclear cells; PPV: Personalized peptide vaccination; PSA: Prostate specific antigen; PSADT: Prostate specific antigen doubling time.K. Itoh is a consultant/advisory board member in Green Peptide Co. A.Yamada is a part-time executive of Green Peptide Co. No potential conflicts of interest were disclosed by other authors.NM conceived of the study, and participated in its design and coordination and drafted the manuscript. KI and AY participated in its design and helped to draft the manuscipt. FM, SS, RO performed the clinical trial and collected the data. SM and TS carried out the immunoassays. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2407/13/613/prepub