Functional Capacity (FC) is a multidimensional construct within the activity domain of the International Classification of Functioning, Disability and Health framework (ICF). Functional capacity evaluations (FCEs) are assessments of work-related FC. The extent to which these work-related FC tests are associated to bio-, psycho-, or social factors is unknown. The aims of this study were to test relationships between FC tests and other ICF factors in a sample of healthy workers, and to determine the amount of statistical variance in FC tests that can be explained by these factors.A cross sectional study. The sample was comprised of 403 healthy workers who completed material handling FC tests  and static work FC tests (overhead working and standing forward bend). The explainable variables were; six muscle strength tests; aerobic capacity test; and questionnaires regarding personal factors , psychological factors , and social factors . A priori construct validity hypotheses were formulated and analyzed by means of correlation coefficients and regression analyses.Moderate correlations were detected between material handling FC tests and muscle strength, gender, body weight, and body height. As for static work FC tests; overhead working correlated fair with aerobic capacity and handgrip strength, and low with the sport-index and perception of work. For standing forward bend FC test, all hypotheses were rejected. The regression model revealed that 61% to 62% of material handling FC tests were explained by physical factors. Five to 15% of static work FC tests were explained by physical and social factors.The current study revealed that, in a sample of healthy workers, material handling FC tests were related to physical factors but not to the psychosocial factors measured in this study. The construct of static work FC tests remained largely unexplained. Functional Capacity (FC) represents the highest probable level of activity that a person may reach at a given moment in a standardized environment ,2. FC isFunctional capacity evaluations (FCEs) are assessments of work-related FC such as lifting and static work. Numerous researchers have adopted the ICF and support the consideration of ICF domains when interpreting FC test results . FCEs faSeveral theories and models corroborate the multidimensional construct of work-related FC ,8. AccorConstruct validity is the ability of an instrument to measure a construct . Within The aims of this study were to test relationships between FC tests and other ICF factors in a sample of healthy workers, and to determine the amount of statistical variance in FC tests that can be explained by these factors.The strength of expected relationships between material handling FC tests  and static work FC tests (standing forward bend and overhead working) with ICF factors are described as hypotheses 1 to 15 in Table During a two-year period, a total of 403 healthy workers (20–60 years of age) executed a 12-item FCE after written informed consent was obtained and the rights of the subjects were protected . We consThe variables measured in this study were classified according to the ICF model Figure  2,16].,16.2,16]Functional capacity was measured with five FCE tests, selected to cover a range of physical activities: (1) lifting low; (2) overhead lifting; (3) carrying ; (4) standing forward bend; and (5) overhead working (static work tests). These were quantified according to the following:Lifting low: Lifting a plastic receptacle from table to floor five times within 90 seconds as the weight is increased in increments 4–5 times.1) Overhead lifting: Lifting a plastic receptacle from table to crown height five times within 90 seconds as its weight is increased in increments 4–5 times.2) Carrying: Carrying a receptacle with two hands for 20 meters as the weight is increased in increments 4–5 times.3) Standing forward bend: For as long as possible, manipulating nuts and bolts while standing, bent forward 30-60° at the trunk, while wearing a five-kilogram weight around the upper thoracic area.4) Overhead working: For as long as possible, manipulating nuts and bolts at crown height while wearing a one-kilogram wrist weight.5) A detailed description of the FCE test protocol is published elsewhere  and can The endpoint of testing could be achieved in several manners. First, the subject could express the desire to terminate the activity. Secondly, the evaluator could end the test because the subject’s safety is in jeopardy. Tertiary, 85% of the age-related maximal heart rate was attained. The test-retest reliability of healthy subjects is good for lifting low ; overhead lifting ; carrying two handed ; standing forward bend test ; and overhead working  ,18.rp = 0.90) [Handgrip strength was measured by the JAMAR hand dynamometer . Isometric handgrip strength was measured using a protocol where subjects were tested in a seated position with the shoulder adducted and elbow flexed 90°. Forearm and wrist were in the neutral position. In previous studies, the test-retest reliability for handgrip strength , intra-, and interrater reliability were good (ICC = 0.85-0.98) in healthy subjects ,19. The  = 0.90) . Elbow m2max), a submaximal Bruce Treadmill Test was performed [2max was predicted employing the following equation:In order to estimate maximum oxygen consumption (VOerformed . Beginni2max through gasometric measurements [r = 0.99) [This formula predicted 86% of the VOurements . The rep = 0.99) .In order to measure self-reported physical activity associated with work, sport, and leisure, subjects completed the Dutch language version of the Baecke Physical Activity Questionnaire (BPAQ) . AnswersThe questionnaire of psychosocial workload and work-related stress (VBBA) includes the Dutch Language version of Karasek’s job content questionnaire which is based on the demand control model ,12,30-32Workers were classified into four levels of physical workload, according to the Dictionary of Occupational Titles (DOT) including sedentary, light, medium, and heavy work ,35.Perceived health status was measured with the Rand 36-item Health Survey (Rand-36) . In thisAge, gender, body height, body weight and level of education data were culminated using questionnaires.r), Spearman (ρ), or point-biserial correlation coefficients (rpbi). To avoid Type I errors, we used Bonferroni’s correction [r), Spearman (ρ) and point-biserial correlations(rpbi) were interpreted as being strong for significant (Pbonf < .002) correlations when r, ρ, rpbi > 0.75; moderate when 0.50 < r, ρ, rpbi ≤ 0.75; fair when 0.25 < r, ρ, rpbi ≤ 0.50; and low when r, ρ, rpbi ≤ 0.25 [r, ρ, rpbi > 0.75; Pbonf < .002) were determined.Descriptive statistics were used to describe the population characteristics. We investigated whether each of the questionnaires was affected by floor or ceiling effect by recoding variables  in cases the median matched the lowest or highest point of a scale. Two authors assessed normality of distributions utilizing histograms ,40. Missrrection . The vali ≤ 0.25 . The valEach of the FC tests were linearly regressed on the Body function, Participation, Environmental and Personal variables by the minimum Bayesian Information Criterion (BIC), which is strongly consistent in finding the best model and often provides interpretable results for practical purposes ,42. To eA total of 403 workers  were tested. Means, standard deviations, and medians of sample characteristics are depicted in Table . Elbow flexion inter-correlated significantly and strong with elbow extension , shoulder abduction , and handgrip strength . Worrying inter-correlated significant and strong with job security .Table Moderate and fair correlations were found between material handling tests regarding gender, body weight, body height, muscle power, and aerobic capacity Table . Low corFair correlations were ascertained between overhead working with aerobic capacity and handgrip strength. The sport-index and four scales of the perception of work correlated low to overhead lifting. For standing forward bend, all hypotheses were rejected. For overhead working, hypotheses 1 to 3 and 6 were not rejected  and the existence of factors from each component of the ICF. In this study, psychological factors were defined according to the background of an individual’s life and living, and therefore, were indicated as personal factors within the ICF framework and not as an impairment in mental function ,2. PhysiWe recommend researchers to replicate this study in a different sample of healthy workers to analyze the robustness of current observations. Further study into the effect of training muscle strength and aerobic capacity on work-related FC tests in healthy workers is also recommended. The empirical evidence of the current study supports fair correlations of FC tests with aerobic capacity. By contrast, in patients with chronic pain, aerobic capacity does not correlate with FC . The traIn healthy workers, it appears that the construct of material handling FC tests is comprised of the physical factors of muscle strength, aerobic capacity, gender, body height, body weight, sport and physical workloads, but, is not comprised of the psychosocial factors included in this study. The construct of static work FC tests remains largely unexplained. Because of the cross sectional design and the healthy study sample in this study, the results should not be interpreted as predictors for future work performance, nor should they be generalized to patients.FC: Functional capacity; ICF: The international classification of functioning, disability and health framework; FCE: Functional capacity evaluations; ICC: Intraclass correlation coefficient; BPAQ: Baecke Physical Activity Questionnaire; DOT: Dictionary of occupational titles; r: Pearson’s correlation coefficient; ρ: Spearman rho; rpbi: Point-biserial correlation coefficient; BIC: Bayesian information criterion; TSK: Tampa Scale of Kinesiophobia; R2: Adjusted R square; B value: Unstandardized regression coefficient; SE: Standard error; P value: Empirical significant level.The authors declare that they have no competing interests.RS has made substantial contributions to conception and design, acquisition of data and analysis and interpretation of the data, drafting the manuscript and critically revising it with important intellectual content. JHB participated in the design of the study, drafting the manuscript and critically revising it with important intellectual content. HW drafted the manuscript and critically revised it for important intellectual content. RD acquired data, drafted the manuscript and critically revised it with important intellectual content. CvdS drafted the manuscript and critically revised it with important intellectual content. MR has made substantial contributions to conception and design, acquisition of data and analysis and interpretation of the data, drafting the manuscript and critically revised it with important intellectual content. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2474/14/180/prepub


In this study, we aimed to determine the relationship between flow-mediated endothelium-dependent vasodilatation (FMD) and carotid artery intima-media wall thickness (IMT), two surrogate markers of atherosclerosis, in a series of Spanish patients with rheumatoid arthritis (RA) without clinically evident cardiovascular (CV) disease.One hundred eighteen patients who fulfilled the 1987 American College of Rheumatology classification criteria for RA, had no history of CV disease and had at least one year of follow-up after disease diagnosis were randomly selected. Brachial and carotid ultrasonography were performed to determine FMD and carotid IMT, respectively.P = 0.02).Carotid IMT values were higher and FMD percentages derived by performing ultrasonography were lower in individuals with a long duration from the time of disease diagnosis. Patients with a disease duration ≤ 7 years had significantly lower carotid IMT (mean ± SD) 0.69 ± 0.17 mm than those with long disease duration (0.81 ± 0.12 mm in patients with ≥ 20 years of follow-up). Also, patients with a long disease duration had severe endothelial dysfunction (FMD 4.0 ± 4.0% in patients with disease duration from 14.5 to 19.7 years) compared with those with shorter disease duration (FMD 7.4 ± 3.8% in patients with disease duration ≤ 7 years). Linear regression analysis revealed that carotid IMT was unrelated to FMD in the whole sample of 118 patients. However, carotid IMT was negatively associated with FMD when the time from disease diagnosis ranged from 7.5 to 19.7 years (In patients with RA without CV disease, endothelial dysfunction and carotid IMT increased with the duration of RA. The association between FMD and carotid IMT values was observed only in patients with long disease duration. Rheumatoid arthritis (RA) is a chronic inflammatory disease associated with increased incidence of cardiovascular (CV) mortality ,2. This Since FMD constitutes a physiologic assessment of endothelial dysfunction and carotid IMT is an anatomic structural measure of subclinical atherosclerosis, it is logical that FMD might be a more useful diagnostic marker than carotid IMT in the early stages of the disease. In contrast, carotid IMT might be considered in the assessment of CV risk among patients with long-standing RA.No relationship between carotid IMT and brachial artery FMD was found in middle-aged men without a history of CV disease who were considered to be at low or intermediate risk for future CV events based on current risk stratification algorithms . HoweverTaking into consideration all of these factors together, and based on the experience of our group in the study of subclinical atherosclerosis not only in RA but also in other chronic inflammatory rheumatic diseases using ultrasound techniques -20, in tBetween January 2008 and December 2009, a series of 118 patients attending the rheumatology outpatient clinic of Hospital Xeral-Calde, Lugo, Spain, who fulfilled the 1987 American College of Rheumatology classification criteria for RA  and had Quantitative variables are described using means and standard deviations (SDs) and medians and interquartile ranges (IQRs), and qualitative variables are described as numbers and percentages. The relationship between carotid IMT (as a dependent variable) and FMD (as an independent variable) was explored using linear regression, adjusting for gender, age at the time of RA diagnosis and years from RA disease diagnosis to ultrasonographic assessment. To further explore this relationship, we repeated the regression analysis by stratifying patients into quartiles defined by the time from RA diagnosis to ultrasonographic evaluation.P < 0.001). In this regard, carotid IMT values were higher if time from RA diagnosis was longer than its median . With respect to this observation, individuals with disease duration from RA diagnosis ≤ 7 years had significantly lower carotid IMT wall thickness (0.69 ± 0.17 mm) than did those with long disease duration . Likewise, FMD decreased as time from RA diagnosis increased (P < 0.001). As shown in the carotid artery ultrasonographic evaluation, individuals with longer disease duration from disease diagnosis had severe endothelial dysfunction (FMD 4.0 ± 4.0% in RA patients with disease duration between 14.5 and 19.7 years versus 3.3 ± 4.4% in those with disease duration ≥ 20 years) compared with RA patients who had shorter disease duration (FMD 7.4 ± 3.8% in patients with disease duration ≤ 7 years).Patients with RA were stratified into four quartiles according to the time from disease diagnosis to ultrasonographic assessment Table . FollowiP = 0.02). In patients included in this range of disease duration, the higher FMD percentages were associated with thinner  carotid IMT values (Figure To explore the relationship between FMD and carotid IMT, linear regression analysis was performed. This analysis disclosed that carotid IMT was unrelated to FMD endothelium-dependent vasodilatation in the whole sample of 118 patients Table . Howevers Figure .et al. [et al. [A series of cellular, molecular and pathophysiological events occur during the progression of RA and may also be involved first in endothelial dysfunction and later in atherosclerosis . A healtet al.  showed t [et al.  found noet al. [et al.'s study [With regard to carotid ultrasonography, it is known that a common carotid artery IMT ≥ 0.60 mm is a marker of atherosclerosis ,31. In aet al.  showed t's study , carotidet al. [et al. [In line with the above findings, using electron-beam computed tomography to measure the extent of coronary artery calcification, Chung et al.  found thet al. . Since tet al. . In this [et al.  showed t [et al. . In addi [et al.  and earl [et al.  without Of main clinical relevance may be the improvement in endothelial function observed in patients with RA following treatment with TNF-α blockers -40 or riPrevious observations showed no relationship between carotid IMT and brachial artery FMD in middle-aged men at low and intermediate risk of experiencing future CV events . HoweverIn summary, our results reinforce the importance of disease duration in the development of atherosclerosis in patients with RA. Brachial FMD and carotid IMT may indicate distinct and independent stages in the complex pathways leading to accelerated atherosclerosis in patients with RA.CV: cardiovascular; IMT: intima-media thickness; IQR: interquartile range; FMD: endothelium-dependent flow-mediated vasodilatation; RA: rheumatoid arthritis; SD: standard deviation.The authors declare that they have no competing interests.CGJ performed the ultrasonographic studies, participated in the design of the study and helped to draft the manuscript. JL participated in the design of the study and data analysis and helped to draft the manuscript. MAGG made substantial contributions to the conception and design of the study, the acquisition of data and the coordination of the study. All authors read and approved the final version of the manuscript to be published.


Zingiber officinale (ginger). We studied the biological effects of ginger extracts via multiple assays and demonstrated the biofunctions in each platform. Investigations of ginger extracts indicated antioxidative properties in dose-dependant manners on radical scavenging activities, reducing powers and metal chelating powers. We found that ginger extracts processed moderate scavenging values, middle metal chelating levels, and slight ferric reducing powers. The antibacterial susceptibility of ginger extracts on Staphylococcus aureus, Streptococcus sobrinus, S. mutans, and Escherichia coli was determined with the broth microdilution method technique. The ginger extracts had operative antimicroorganism potentials against both Gram-positive and Gram-negative bacteria. We further discovered the strong inhibitions of ginger extracts on lethal carcinogenic melanoma through in vivo xenograft model. To sum up, the data confirmed the possible applications as medical cosmetology agents, pharmaceutical antibiotics, and food supplements.Supercritical fluid carbon dioxide extraction technology was developed to gain the active components from a Taiwan native plant,  Antioxidative components are of importance due to the abilities to reduce free radical-mediated degradations of cells, tissues, and organisms in humans . There aWith the long-term exposure in high oxidative stress, the normal cells often accompany with carcinomas and transfer into sick status. The strategy of diminishing oxidative stress is a good solution to keep human being physiologically healthy. Therefore, natural antioxidants from plant species which decrease oxidative stress from intrinsic and external sources have huge applicable biofunctions in human health care , 4.A worldwide troubling public health issue emerged from hospital-acquired or nosocomial illness is caused by infections microorganisms in recent decades. Pathogenic bacteria are able to survive for extended periods on human superficial skin, mucosa, or environmental surfaces and have been implicated in infectious outbreaks within hospitals, medical facilities, and institutions in many countries . With thHuman skin is normally contacted with damage stress, which is produced by external and intrinsic sources, such as ultraviolet radiation, free radicals, and reactive oxygen species. There are many studies about the ultraviolet radiation which are responsible for skin aging or tumorigenesis. Melanoma, a malignant tumor of epidermal melanocytes, is one of the most deadly skin cancers . During Traditionally, natural matrices have been obtained by means of conventional extraction with organic solvents. Nevertheless, the extraction changes in the physicochemical properties of extracts could alter their functionalities. Thus, the extraction processes should be performed at suitable and mild conditions. Supercritical carbon dioxide extraction is an advanced technology that has many advantages, including low environmental impacts due to no residue of harmful solvents, noncorrosiveness, nontoxicity, and easy separation from extracts , 13. It  Zingiber officinale Roscoe , is used widely as a spice throughout the world. In Chinese medicine, ginger has been used traditionally as a treatment for allergy, constipation, asthma, diabetes, nervous diseases, rheumatism, toothache, stroke, and antimicroorganism infection [Ginger, the powdered rhizomes of the herbnfection . In addinfection –18. The nfection . The preDimethyl sulfoxide (DMSO) and Luria-Bertani broth were purchased from Sigma-Aldrich Chemical Inc. . Dulbecco Modified Eagle Medium (DMEM), fetal bovine serum (FBS), penicillin, and streptomycin were purchased from Life Technologies CO., Ltd. . The plant specimen was authenticated by Dr. Chih-Chen Lee, Derlin Biotech Corporation , where voucher specimens were kept. The air-dried extracts of ginger were cultivated and obtained in the district of Nantou, Taiwan (2012). All buffers and other reagents were of the highest purity commercially available.The supercritical carbon dioxide fluid extraction was performed on United States supercritical fluid extractor  with the extractor volume of 5.0 L. The liquid ethanol from a cylinder was pressured to reach the supercritical state with a piston pump and cooling at −4°C in a water bath before it passed into the extraction vessel (with 5.0 L inner extraction container). The extraction pressure was adjusted by needle and micrometric valves. The extraction temperature was controlled by a thermostatic batch. The flow rate of ethanol was regulated by a rotameter. The container was placed into the extraction vessel, and the consumption volume of ethanol was recorded by a gas meter. After the temperature of the extraction vessel reached the set point, the micrometric valves were closed. Then, ginger extracts (500 g) were loaded into a 5.0 L stainless steel extraction container (9.2 cm ID) with stainless steel filters placed at both ends to prevent carryover of the particles. The pressure of extraction vessel was controlled at 5,800 psi by a needle valve and set at 2 hours of static extraction time with a timer. The solute-rich fluid departing from the extractor was expanded through micrometric valves to atmospheric pressure. The flow rate was set at 6 NL/min and the total volume of gas was measured with a gas meter. The carbon dioxide discharge was recycled to cultivate ginger extracts to reduce carbon dioxide emission to the environment.μg/mL) were added to 1 μL of stable DPPH (60 μM) solution and measured with a 96-well plate. When DPPH radicals react with antioxidative agents donating hydrogen, the solution color is reduced resulting in a decrease in absorbance at 517 nm , and DPPH solution changes its color to light yellow when free radicals are scavenged. Various concentrations of ginger extracts (100~5000 t 517 nm . The anaμg/mL) was investigated according to a previously described method [2·4H2O (10 μL). The reaction was initiated by the addition of 5 mM ferrozine (20 μL), and the mixture was vigorously shaken and left standing at 25°C for 10 min. The absorbance of the mixture was read at 562 nm against a blank. EDTA was used as a positive control, and the chelating activity calculation formula was similar to  were determined according to the method of [μL of 67 mM phosphate buffer (pH 6.8) and 2.5 μL of 20% K3Fe(CN)6. The mixture was incubated at 50°C for 20 min, and then 160 μL of trichloroacetic acid (10%) was added to the mixture to centrifuge for 10 min at 3,000 ×g. The upper layer of the solution (75 μL) was mixed with 2% FeCl3 (25 μL), and the absorbance was assayed with a 96-well plate at 700 nm. A higher absorbance demonstrates a higher reductive capability. BHA was used as a positive control.The reducing powers of ginger extracts (100~5000 ethod of . In brieStaphylococcus aureus (ATCC 29213), Streptococcus mutans (ATCC 25175), S. sobrinus (ATCC 33478), and Escherichia coli (ATCC 35218). The most common species of staphylococcus is S. aureus, which causes staphylococcus infections and is frequently found in the human respiratory tract and on the skin surfaces. The emergence of antibiotic-resistant forms of pathogenic S. aureus  is a worldwide problem in clinical medicine [Streptococcus species is S. sobrinus which is a spherically shaped anaerobic and Gram-positive bacterium. They grow in pairs or chains, and they are not motile and do not form spores. S. mutans is a facultatively anaerobic, Gram-positive, coccus-shaped bacterium commonly found in the human oral cavity and is a significant contributor to tooth decay. The most intensively studied prokaryotic model organism is E. coli, a Gram-negative, rod-shaped bacterium commonly found in the lower intestine of warm-blooded organisms. This bacterium is easily and inexpensively grown in a laboratory setting and has been studied intensively in the past half century [The four microorganismic strains used within this study, which were purchased from American Type Culture Collection (ATCC), included medicine . Another century .5 CFU/mL microbial suspensions of four bacteria, S. aureus, S. mutans, S. sobrinus, and E. coli, were incubated in each well at 37°C for 24 hours. The microbial bacteria were then harvested in normal saline and adjusted to McFarland 0.5 (1.5 × 108 CFU/mL). One milliliter of each bacterial sample suspension was centrifuged at 9,000 rpm for 5 minutes and then treated with 0.3 mL of an extract to obtain a final concentration of 10% v/v. Reactions were compared at 25°C at intervals of 5, 30, 60, 180, 300, and 900 seconds, respectively. A well containing DMSO was used as the growth blank vehicle control; a well containing medium only was used as the negative control; all other wells contained the experimental groups treated with ginger extracts. The examination was considered valid if the well for the growth control group was positive and those for other groups were negative. After the specified reaction times, the bacteria were centrifuged for 2 minutes, washed once, and suspended in sterile saline water. A 104-fold dilution of the bacterial suspension (100 μL) was then plated on blood agar plates. After 24-hour incubation period time, the bactericidal effects of the extracts were determined in each sample by measuring bacterial growth in cultures. The inhibition of bacterial growth was then measured by comparison with normal growth observed in microbes not treated with the testing samples.The antimicrobial properties of the ginger extracts were investigated using previously described methods. Briefly, 103 cells/well. The medium was then changed, and cells were maintained in either solvent alone (control cells) or in the presence of the indicated ginger extracts (50~1000 μg/mL) in a final volume of 100 μL within 10% FBS cellular culture medium. Each sample was added to a microplate and incubated under the same conditions as aforementioned.The human skin cancer A375 cell line was purchased from the Bioresource Collection and Research Center . This malignant melanoma A375 cell line was derived from a 54-year-old female. The cells were seeded in 96-well plates at a density of 7 × 10In this study, the use of animals complied with the Guiding Principles in the Care and Use of Animals of the American Physiology Society and was approved by the National Kaohsiung Medical University and Use Committee. BALB/c nu/nu female mice (4-5 weeks) were purchased from BioLASCO Experimental Animal Center . The mice were housed in Plexiglas cages in a temperature-controlled room (22 ± 1°C), on a 12-hour/12-hour light/dark schedule, and with free access to food and water. After one week, ten mice were randomly divided into 2 groups, control and drugs treatment groups. in vivo experiments were performed at the animal center . Mice were implanted subcutaneously with 1 × 107 of A375 cells in 0.1 mL PBS injected subcutaneously in each mouse. Mice were treated four times a week with a subcutaneous injection of ginger extract (300 mg/kg) until sacrifice at day 35. The diameters of xenograft tumor were measured at 4 days intervals with vernier calipers and calculated as length × width2/2 in mm3 [The performance of xenograft tumor assay was described previously with minor modifications . In brie2 in mm3 . Mice used the tape fixed under test location and the moorLDI2 laser Doppler blood perfusion imagers  was used to measure in the tumor periphery, and the digital color-coded images were analyzed to quantify blood flow in the back .t-test for paired values.The data were expressed as the mean value obtained in three experiments. Statistical comparisons were performed by Student μg/mL to 5,000 μg/mL). DPPH free radical scavenge testing mechanism is an acknowledged system by which antioxidants act to inhibit oxidation products; hence, this DPPH scavenging platform has been widely applied as one of the indicators for antioxidant abilities. In this DPPH assay procedure, antioxidants are able to decrease the stable purple DPPH radicals to the light yellow colored diphenyl-picrylhydrazine. The inhibition values of ginger extracts were listed in μM was as a positive control within this assay. It showed a dose-dependent manner that ginger extracts had moderate-high scavenging capability in DPPH assay. The following purpose of this section was to survey the antioxidative properties of ginger extracts from supercritical fluid carbon dioxide extraction, and the testing samples were added at various concentrations , and the positive control was BHA at 100 μM (1.92 ± 0.04). Ferric reducing antioxidant power assay is a simple and reliable platform, measuring the reducing potential of an antioxidant reacting with a ferric 2,4,6-tripyridyl-s-triazine Fe(III)-TPTZ complex, producing a dark blue colored ferrous Fe(II)-TPTZ complex by an adopted reductant. This complex has a conspicuous blue color which can be monitored at 700 nm. A higher absorbance in spectrogram indicates a higher ferric reducing power. In S. aureus, S. sobrinus, and E. coli, but not S. mutans. The antibacterial assay illustrated that ginger extracts effectively inhibited both Gram-negative and Gram-positive bacteria. Generally, for each microbe, whether Gram-negative or Gram-positive, the growth phenomenon was time-dependently inhibited by the exposures of 5, 30, 60, 180, 300, and 900 seconds. The next experiment was to compare the antibacterial effects of ginger supercritical carbon dioxide fluid extracts. in vivo tumor xenograft by treating BALB/c nu/nu female mice bearing human malignant melanoma A375 cell lines, at a concentration of 300 mg/kg. After the establishment of palpable tumors (the mean tumor volume was around 50~100 mm3), animals received subcutaneous injections of ginger extracts four times for one week. After 35 days, we found out that the cellular A375 proliferation was reduced significantly compared to the vehicle control, and photographs in The cellular proliferation assay illustrated antigrowth effects in nude mice on skin cancer cells . In thisWithin mice receiving the treatment regimens, no gross signs of toxicity were observed  Figures . In the E. coli and S. sobrinus) and gram-positive bacteria, (S. aureus and S. mutans), but S. mutans has no obvious effect in antibacterial activities. In animal experiments, the nude mice were pretreated with A375 melanoma cells, and the ginger extracts illustrated an obvious reduction in cell proliferations in vivo xenografts. To sum up, it was the first study of ginger extracts from supercritical fluid carbon dioxide to be potentially applied in food additive, infectious inhibition, or anticancer agents.Our research results showed that ginger extracts from supercritical fluid carbon dioxide extraction demonstrated DPPH free radical scavenging ability, reducing power and chelating property in dose-dependent manners. This research directly confirmed that the importance of ginger extracts had positively influencing levels of dietary antioxidants in the human body. Ginger extract proved the antibacterial activities against both gram-negative bacteria (


Stichopus horrens flesh was explored as a potential source for generating peptides with angiotensin-converting enzyme (ACE) inhibitory capacity using 6 proteases, namely alcalase, flavourzyme, trypsin, papain, bromelain, and protamex. Degree of hydrolysis (DH) and peptide profiling (SDS-PAGE) of Stichopus horrens hydrolysates (SHHs) was also assessed. Alcalase hydrolysate showed the highest DH value (39.8%) followed by flavourzyme hydrolysate (32.7%). Overall, alcalase hydrolysate exhibited the highest ACE inhibitory activity  followed by flavourzyme hydrolysate , trypsin hydrolysate , papain hydrolysate , bromelain hydrolysate , and protamex hydrolysate . The SDS-PAGE results showed that alcalase hydrolysate represented a unique pattern compared to others, which yielded potent ACE inhibitory peptides with molecular weight distribution lower than 20 kDa. The evaluation of the relationship between DH and IC50 values of alcalase and flavourzyme hydrolysates revealed that the trend between those parameters was related to the type of the protease used. We concluded that the tested SHHs would be used as a potential source of functional ACE inhibitory peptides for physiological benefits. Hypertension, the leading cause of cardiovascular disease in the world, is one of the most common disease affecting humans . It is aAngiotensin-converting enzyme (ACE) which is responsible for the elevation of blood pressure acts as an exopeptidase that converts an inactive form of decapeptide (angiotensin-I) to a potent vasoconstrictor, an octapeptide (angiotensin-II), and inactivates the catalytic function of bradykinin, exhibiting depressor action , 2. PeptAcaudina molpadioidea gelatine hydrolysate  wμL of the derivatized sample was injected into the HPLC system equipped with photodiode array detector . A C18-reversed phase column  maintained at 43°C was used for separation purposes. The mobile phase consisting of buffer A  and buffer B  was flushed through the column at a flow rate of 1 mL/min using a linear gradient system. The UV absorption detector at a wavelength of 254 nm was employed to monitor amino acids. The identification of unknown amino acids was based on the comparison of their retention times with those of pure standards, whereas for quantification purposes standard calibration curves were used. The results were analyzed and computed by the Borwin chromatography software .Amino acid composition was studied using a reversed-phase high-performance liquid chromatography (HPLC) system . A WaterS. horrens hydrolysate samples were mixed 1 : 1(v/v) with the loading dye. The stacking gel concentration was 4% and resolving gel 15%. Gel was run at 30 mA. Protein bands were stained by silver nitrate kit (Sigma Aldrich).SDS-PAGE was performed according to the method of Laemmli . BrieflyProtein concentration was determined following the method of Bradford using micro protein kit from Sigma Aldrich.h) to the total number of peptide bonds in the substrate studied (htot). In each case, calculation was based on the amount of NaOH or HCl added to maintain the pH constant during the hydrolysis using the following equation [B is the amount of acid or base consumed (mL) to keep the pH constant during the hydrolysis. Nb is the normality of acid or base, MP is the mass (g) of protein (N × 6.25), and α is the average degree of dissociation of the α-NH2 groups released during hydrolysis expressed aspK values were calculated using the equation below [T is the hydrolysis temperature in Kelvin. The degree of hydrolysis (DH) is defined as the percent ratio of the number of peptides dissociated (equation :(2)DH  on below (4)pK=7.htot) in sea cucumber hydrolysate was calculated 3.44 meq/g. The total number of peptide bonds  at S. horrens as the substrate of the proteolysis was measured. The result of proximate composition of S. horrens for protein, moisture, fat and ash contents was as 2.83, 93.10, 0.21 and 2.70 g/100 g (fresh weight), respectively. The protein content (2.83 g/100 g) determined in this study was lower than that reported for other species of sea cucumber namely Cucumaria frondosa and Holothuria tubulosa [Parastichopus parvimensise, Parastichopus californicus [Acaudina molpadioides [Holothuria tubulos [Apostichopus japonicus [The protein content of tubulosa . Meanwhitubulosa . The ashfornicus , Acaudinadioides , Holothu tubulos , and Apoaponicus . 50 value or not. Hence, the DH values of sea cucumber hydrolysates were analyzed and presented in P ≤ 0.05) higher than flavourzyme.In order to produce ACE inhibitory peptides, sea cucumber flesh was hydrolysed with 6 different proteases. Proteases degrade protein and produce peptides with various lengths and sequences. Degree of hydrolysis (DH) is therefore an indicator of such hydrolysis progress which reflects the ability of proteases to access available cutting sites of the protein. DH, thereby, is defined as the proportion of peptides bond cleaved during proteolysis to the total peptide bond available in the protein structure. DH, in the present study, was determined for two purposes: (a) to find out the trend of DH values over the hydrolysis time; (b) to have an idea wheather the DH changes during time course can reflect the variation in ICBacillus licheniformis NH1 reported for seafood protein like, sardine [Salmo salar [S. horrens is degradable by all six proteases, and the differences among their DH values imply that the number of available cutting sites for alcalase is much higher than the others. The presently recorded asymptotic curves of alcalase and flavourzyme are similar to other studies using flavourzyme and crude extract enzymes from  sardine , pacific sardine , sardine sardine , threadf sardine , Salmo smo salar , cuttlefmo salar , and cutmo salar . However50 over time, S. horrens was hydrolysed using different proteases for up to 300 min  to 0.41 mg/mL (at 300 min of hydrolysis), indicating highest ACE inhibitory activity. However, IC50 for the other enzyme hydrolysates over the time course of 300 min hydrolysis varied from 2.47 to 1.49 mg/mL (trypsin), 5.26 to 1.56 mg/mL (bromelain), 2.24 to 0.78 mg/mL (flavourzyme), 3.57 to 2.31 mg/mL (papain), and 6.38 to 3.42 mg/mL (promatex). An overall decrease in IC50 value with the increase of hydrolysis time reflects that hydrolysis is effective towards enhancing ACE inhibitory capacity of the hydrolysates. In order to study the change of IC 300 min . Hydroly50 over time can be explained from two aspects: changes in the number of amino acids in peptide chain and most importantly the amino acid residues presented as N- or C-terminal sequence of peptide. Small peptides with certain amino acids in their N- and C-terminal sequences are shown to exhibit high ACE inhibitory effect [50 values of hydrolysates could have resulted from the changes occurred in the two terminals of generated peptides, the number of amino acids and their proportions in the crude hydrolysates at any time. Alcalase degrades peptide bond of aliphatic or aromatic amino acids such as Leu, Phe, Tyr and Trp [Variation in ICy effect . It was y effect , 34. They effect , 35. Gen and Trp . While t and Trp .50 were lower than those reported for some other marine hydrolysates like oyster, scallop, codfish skin, and herring skin (>10 mg/mL) [50 values of whole bovine plasma hydrolysates produced by alcalase and papain  [50 values of albumin hydrolyzed with alcalase and casein degraded by trypsin was quite close to that determined presently for alcalase hydrolysate of S. horrens. IC50 value of 0.615 mg/mL for the fraction containing peptides of molecular weight lower than 2 kDa from Acaudina molpadiodea has been reported [Brachionus rotundiformis, using different proteases , alcalase produced a mixture of peptides exhibiting IC50 value of 0.63 mg/mL [Bombyx mori showed an IC50 value of 0.697 mg/mL which was close to the one from Alaska Pollack skin hydrolysate [50 value of alcalase hydrolysate of Spirulina platensis  was 0.47 mg/mL [S. horrens flesh and that resulting mixture of peptides was more efficient towards inhibition of ACE.Several researchers have studied the applications of different proteases on various sources to investigate their ability for producing ACE inhibitory peptides , 39. How0 mg/mL)  but high0 mg/mL) . The IC5, resp.)  were mucreported . Likewis63 mg/mL . Similarrolysate , while I47 mg/mL . These r50 values depend on both size and N- and/or C-terminal sequences of the peptide. Therefore, it is crucial to investigate any relationship that might exist between DH and IC50  exists. Plotting DH and IC50 revealed that despite an elevating DH value during the time-course of hydrolysis, the two hydrolysates represented a different pattern concerning IC50 values. The alcalase hydrolysate IC50 value showed a downward trend whereas flavourzyme hydrolysate IC50 value exhibited mixed pattern trend with two phases: an increase in DH value up to 20% while IC50 decreased, but by further increase in DH value again IC50 values increased. Alcalase hydrolysate showed an almost steady decrease in IC50 values with the progression of hydrolysis. Balti et al. (2010) reported a correlation coefficient of 0.83 between DH and ACE inhibitory activity of cuttlefish muscle hydrolysate prepared using several proteases [50 or inhibitory effect might not be correlated in certain cases. DH indicates the progress of proteolysis and generation of small peptides while ICand IC50 . Both hyS. horrens hydrolysaes during the proteolysis time was investigated using SDS-PAGE. S. horrens due to the cleavage of nearly all proteins in the high molecular weight region and generation of polypeptides below 20 kDa. This result is in line with the DH and another finding of this study which indicates that alcalase exhibits the lowest IC50 value (0.4 mg/mL) compared to the other enzymes. Collagen has been found to be the major protein compound of sea cucumber consisting of α chain with the molecular weight of 100 kDa [Since the size of the generated peptides is crucial on the ACE inhibitory effect as previously reported , 34, the 100 kDa , 45. The 100 kDa .S. horrens protein could possess the most cutting sites suitable for alcalase in comparison with papain and trypsin enzymes.  On the other hand, papain and trypsin were almost effective towards degrading most of the large protein components most probably including collagen since from the first sample (taken at 30 min of proteolysis) large protein bands (116 kDa) nearly disappeared. The protein range of hydrolysate produced by alcalase, papain and trypsine was below 20, 27, and 34 kDa, respectively. Such lower molecular weight peptides generated by alcalase indicates that The electrophoretic patterns of the other enzyme hydrolysates were found to be entirely dissimilar and other hydrolysates mostly contained peptides with molecular weight in the range between 4 and 116 kDa. As for hydrolysates prepared by flavourzyme and protamex, protein bands between 34 to 66 were disappeared before 300 min of hydrolysis. In the case of bromelain, protamex, and flavourzyme hydrolysates, the protein bands around 34 kDa were almost hydrolyzed within 5 h. The intensity of the protein bands below 14 kDa, in the case of flavourzyme, increased as the hydrolysis time reached to 300 min. Papain and trypsin were more effective towards degrading most of the larger proteins compared to flavourzyme, protamex, and bromelain. Interestingly, the most marked reduction in the molecular weight of peptide was found in alcalase hydrolysate whose protein intensity was quite high (6 to 20 kDa). Our results were in agreement with those of some previous studies conducted on different substrates, that is, Atlantic cod viscera  and red S. horrens and enzyme-treated S. horrens  hydrolysates, calculated on dry weight basis, are given in Stichopus japonicas [H. scabra, H. nobilis, H. impatiens, H. multipilula, Actinopyga echinites, and Thelenota ananas [ The contents of different amino acids of untreated aponicas . Glutamia ananas . A simil50 values. Alcalase hydrolysate possessed the lowest IC50 value followed by flavourzyme, trypsin, and bromelain hydrolysate suggesting that position of those amino acids in the peptide sequence is more crucial than their total amounts.As mentioned before, it is known that hydrophobic and positively charged amino acids existing in peptide C-terminal contribute to the peptide inhibitory properties. Flavourzyme hydrolysate exhibited the highest hydrophobic content followed by alcalase and trypsin hydrolysate. In case of positively charged amino acids , almost the same pattern happened; however, it is not comparable with those of ICS. horrens have good potential for production of ACE inhibitory peptides and also offered high amounts of valuable amino acids. Of the different proteases tested, alcalase was found to be the most efficient for production of hydrolysate with the best ACE inhibitory activity . Therefore the belief of sea cucumber having antihypertensive effect in the folk medicine is supported in vitro. Thus, S. horrens protease hydrolysates could be employed as a potential source of functional ACE inhibitory peptides suggesting their uses for regulating normal blood pressure in hypertensive humans, in addition to obtaining other physiological benefits. This study revealed that enzyme hydrolysates of


Trichomonas vaginalis isolates recently. The objective of this study was to determine the minimum lethal concentration (MLC) of metronidazole in resistant and sensitive strains, as well as genetic patterns of these stains by PCR method.Metronidazole is drug of choice recommended by WHO for treatment of trichomoniasis, however, some reports claims drug resistance in Trichomonas strains (resistant and sensitive) tested by standard method. Finally, all sensitive and resistant strains examined by PCR technique.From February 2006 to March 2007, in a cross sectional study, clinical and wet mount examination of vaginal smear along with culture were performed on 683 women attending to public and private outpatient clinics in Hamadan. Trichomoniasis marked based on major clinical symptoms. Diagnosis confirmed using wet mount microscopically and culture in Diamond medium. A serial concentration of metronidazole was provided and all isolated T. vaginalis by wet smear and culture. The minimum lethal concentration (MLC) for clinically sensitive isolates was 25 µg/ml; however, this concentration for resistant isolates was 200 µg/ml after 24 h and 100 µg/ml after 50 h. The results of PCR examination of DNA from sensitive and resistant isolates had same pattern. The lanes appeared by two primers were 98 bp and 261 bp for both clinically sensitive and resistant strains.Only 15/683, (2.2%) of patients clinically diagnosed trichomonal vaginitis were positive for T. vaginalis has not relation to genetic variations and might be related to some physiologic pathways of organism.Resistance to metronidazole in  Trichomonas vaginalis and also other important intestinal protozoa, such as Giardia lamblia and Entamoeba histolytica in the world and has high prevalence and incidence between all STD, even in the developed countries such as US. Its prevalence strongly related to various cultural and social norms in different societies, in relation to sexual partnership, monogamy or polygamy etc. Metronidazole is an antimicrobial agent that has been used for more than 50 years for the treatment of infection caused by tolytica. Metronind so on , 3. TreaGardnerella vaginalis of metronidazole against T. vaginalis. The patients confirmed with trichomoniasis received one standard dose of metronidazole (2g single dose) and treatment repeated one more dose if the complaints were continued and parasite were detected again by same methods. Resistant cases defined as “cases in which two standard courses of treatment fails to cure clinically and parasite detect by above-mentioned methods”  were taken from all with vaginal infection signs. Primarily, direct wet mount examination was done and another specimen was cultured in Diamond medium for methods” .T. vaginalis isolates (n = 10), susceptible to metronidazole clinically, and five drug-resistant isolates of parasite were used in this study. These wild-type strains were isolated from female patients examined at the University Women Hospital and in a private clinic. The symptomatic infection of most patients was successfully cured with a single course of metronidazole and drug susceptibility of the isolated strains was confirmed by an in vitro assay for MLC test.Minimum lethal concentration (MLC) of metronidazole  was done for all isolated parasites by method described by Upcroft and Upcroft, 2001 . T. vagiAll parasites isolated from symptomatic patients were grown in culture medium. The parasites were sub-cultured three times a week in the Research Laboratory of Department of Parasitology & Mycology. Parasites to be used in drug susceptibility assays were grown for 1 day more following regular sub-culturing and were in the log phase of growth.2 (This experiment was done in the condition of aerobic assay. Stock solution (0.1 M) of drug (200 mg/ml metronidazole [Sigma]) in dimethyl sulfoxide (Sigma) were prepared and stored at -20°C until use. Metronidazole purchased in powder stock and was dissolved in DMSO 100%; drug went into solution in DMSO difficulty. To obtain better solubility, solution was autoclaved at 121° C for 15 min at15 lb/inThe stock solution was diluted in medium to the research required concentrations. A useful starting concentration was 400µM, which yielded a maximum concentration in the assay of 100µM (20µg of metronidazole per ml).Drugs were serially diluted, with dilutions ranging from 400 µg/ml to 3µg/ml. Plates were then incubated at 37°C for 48 h and read with an inverted phase-contrast microscope . The micro plate titration well with the lowest concentration of drug in which no motile trophozoites were seen was reported as the MLC. The end point was confirmed by failure of non-motile parasites to grow after re-inoculation into drug-free medium.T. vaginalis cultures 10x5 trophozoites per well were required for aerobic assays.For each isolate and drug concentration, there were four replicates with drug and two without drug. In this study, for According to Upcroft and Upcroft method (2001), the plate aerobic assay mixtures were placed directly in a non-gassed incubator. Trophozoite growth was monitored daily by comparing control and drug-containing wells in the same row using an inverted microscope. Trophozoite numbers were scored using 1+ , 3+ , and 4+ (a confluent well) .Genomic DNA was extracted from specimens by phenol-chloroform-isoamyl alcohol method. A PCR assay was done by following primers :TVK3: AT TGT CGA ACA TTG GTC TTA CCC TC, and TVK7: TCT GTG CCG TCT TCA AGT ATG CT. vaginalis–specific repeat DNA fragment. The confirmatory assay used primers TVA5-1: AT GTT CTA TCT TTT CAT TGT and TVA6: GAT CAC CAC CTT AGT TTA CA to amplify a 98-bp fragment of a T. vaginalis specific sequence  were found infected by  product . AccordiT. vaginalis were shown for the first time in Iran. All isolates investigated, including metronidazole-resistant isolates, were susceptible to metronidazole, with different concentrations. The lowest effective concentration of metronidazole was 20µg/ml; however, the susceptibility of the isolates was dependent on exposed time. No significant differences in metronidazole susceptibilities were observed between metronidazole-susceptible and metronidazole-resistant isolates.In the present study, different concentrations of metronidazole on clinically susceptible and resistant isolates of (nim) conferring resistance to nitroimidazoles have been isolated in different genera of gram-positive and gram-negative anaerobic bacteria, including Bacteroides species  of Iranian isolates which may be related to metronidazole resistance (T. vaginalis from high risk population in Iran but, did not present any resistance evidence in the isolates (T.vaginalis infection and the degree of resistance in the parasite indicate a need for a non-nitroimidazole treatment for refractory trichomoniasis, such as miltefosine (inations –22; howeinations  or in soinations . Kazemi sistance . Althougisolates , 27. Inctefosine . The cur


At recurrence the use of nitrosoureas is widely-used as a therapeutic option for glioblastoma (GBM) patients. The efficacy of fotemustine (FTM) has been demonstrated in phase II clinical trials; however, these papers report a wide range of progression-free-survival (PFS-6 m) rates, ranging from 21% to 52%. We investigated whether FTM could have a different response pattern in respect to time to adjuvant temozolomide failure, or whether specific independent risk factors could be responsible for the wide range of response rates observed.Recurrent GBM patients have been treated with fotemustine 75-100 mg/sqm at day 1, 8, 15 and after 4/5 weeks of rest with 100 mg/sqm every 21 days. Patients were stratified in 4 groups according to time to temozolomide failure: before starting (B0), during the first 6 months (B1), after more than 6 months of therapy (B2), and after a treatment-free interval (B3). Primary endpoint was PFS-6 m. A multivariable analysis was performed to identify whether gender, time after radiotherapy, second surgery and number of TMZ cycles could be independent predictors of the clinical benefit to FTM treatment.163 recurrent GBM patients were included in the analysis. PFS-6 m rates for the B0, B1, B2 and B3 groups were 25%, 28%, 31.1% and 43.8%, respectively. The probability of disease control was higher in patients with a longer time after radiotherapy (p = 0.0161) and in those who had undergone a second surgery (p = 0.0306).FTM is confirmed as a valuable therapeutic option for patients with recurrent GBM and was active in all study patient groups. Time after the completion of radiotherapy and second surgery are independent treatment-related risk factors that were predictive of clinical benefit. Glioblastoma (GBM) is the most common malignant primary brain tumor in adults and is associated with a poor prognosis . RadiothNovel target therapies, including antiangiogenic drugs, are under investigation, but the role of such drugs in the treatment of GBM is still being debated . ResearcRechallenge with TMZ is often used following GBM recurrence, but its usefulness is questionable due to conflicting data -9. RecenFotemustine (FTM) is a third-generation chloroethylnitrosourea containing a phosphoalanine carrier group attached to the nitrosourea radical . This chBased on these assumptions, the aim of the present retrospective analysis was to assess whether FTM could have a different response pattern compared to the time to adjuvant TMZ failure, or whether specific independent risk factors could be responsible for the wide range of survival rates observed.Six Italian sites participated in this retrospective pooled analysis. The study was done in accordance with the provisions of the Declaration of Helsinki and local regulations. The study was approved by the institutional review board at each study centre.Adult patients with recurrent or progressive, histologically-confirmed GBM who underwent surgery and the Stupp regimen (radiotherapy plus concomitant and adjuvant temozolomide)  were incContrast-enhanced (gadolinium-DTPA 0.2 mmol/kg) MRI of the brain was uniformly adopted for tumor assessment and response evaluation. Baseline MRI examination was performed before administration of FTM, and subsequent evaluations were carried out after completion of the induction phase, every two cycles during the maintenance phase.Glioblastoma patients were stratified into three groups according to time to TMZ failure, as proposed by Perry and colleagues10. Group B1 (early): progression while receiving adjuvant TMZ before completion of six cycles of adjuvant TMZ. Group B2 (extended): progression while receiving extended adjuvant TMZ beyond the standard six cycles, but before completion of the adjuvant treatment. Group B3 : progression after completion of TMZ.http://ctep.cancer.gov/forms/CTCAEv4.pdf).We added a fourth group, B0: progression after radiotherapy completion and before initiation of adjuvant TMZ. Response was assessed clinically and radiologically using evaluated with Macdonald’s criteria . Safety The primary endpoint of the study was evaluation of progression-free survival at six months (PFS-6 m) according to time of TMZ failure .Secondary endpoints were evaluated in all four patient groups, plus the whole population, and were: response rate (RR), disease control (DC), overall survival at 1 year (OS-1y) and time to progression (TtoP). Responses were confirmed as complete (CR), partial (PR) and stable (SD). RR was defined as CR + PR, otherwise DC is defined as CR + PR + SD. OS was measured from the start of FTM to death for any reason, or last follow-up assessment. Disease progression (DP) was confirmed with two consecutive radiologic investigations.A multivariable analysis of the whole population was performed to identify whether gender, time after radiotherapy, second surgery and number of TMZ cycles could be independent predictors of clinical benefit (DC) to the FTM treatment. The multivariable model was built adopting the so-called epidemiological approach, which consists of selecting covariates regardless of univariate findings or statistical elimination methods  but depending only on their expected or potential clinical importance. Time to progression and time to death, in the whole population, was estimated by Kaplan-Meier analysis between responder and non-responder patients.The demographic and clinical-pathological characteristics were summarized using descriptive statistics. In general, absolute and relative frequencies were employed to summarize qualitative variables, while arithmetic mean, standard deviation (SD), range, median and interquartile range were used to summarize quantitative data.Penalized multivariable logistic regressions with gender, time from radiotherapy, second surgery, and number of TMZ cycles as covariates were performed adopting the Firth’s correction in order to adjust estimates for potential over-fitting, skewed data and multicollinearity. Results were reported as adjusted  odds ratios (ORs) with an associated 95% CI and two-tailed probability values. Statistical calculations were carried out using SAS version 9.2. A two-tailed P-value of 0.05 was used to define statistically significant results.One hundred and sixty-three patients with a diagnosis of recurrent GBM were included in the analysis. All patients followed a Stupp regimen as first line treatment; all patients were treated with a combination of radiotherapy and TMZ, 87.7% 143/163) received adjuvant TMZ, and 12.3% (20/163) of patients experienced a recurrence immediately following the conclusion of radiotherapy and were thus evaluated as the B0 group. Patient characteristics are listed in Table 43/163 reAll patients received at least one dose of the study drug, with a median number of 4 administrations up to a maximum of 20 doses. During the induction phase, 100 mg/m2 of FTM was the median administered dosage (range 75–100).Response to FTM treatment was documented in all patients Table . The PFSRR was reported as 15.0%, 10.0%, 15.6% and 16.7% of the B0, B1, B2 and B3 groups respectively. The DC rate ranged from 36.0% (B1) to 66.7 (B3), while OS-1y ranged from 25.0% (B0) to 31.3% (B3).The multivariable logistic regression evaluated the impact of gender, time from radiotherapy, second surgery, and number of TMZ cycles on DC in the whole population Table . The proFTM administration was well tolerated and the most relevant grade 3–4 toxicity events were leucopenia (6.7%) and thrombocytopenia (9.8%), as expected. No differences in tolerability were observed between study groups  were those with the lowest clinical response for all study endpoints: PFS-6 m (25.0%), OS-1y (25.0%), and RR (15.0%).A comparison between our data and those reported by Perry could suggest a potentially different response pattern between recurrent GBM patients treated with FTM and those with TMZ, in respect to adjuvant TMZ failure. TMZ is active in early and late progression patients, while FTM was always active in recurrent patients: the PFS-6 m, OS-1y, RR and DC rates are reported in Figure Hypothetically, the different response pattern between patients treated with FMT and those with TMZ could be due to the different mechanism of action between the two alkylating agents. FTM, unlike TMZ, a known mono-functional DNA methylating agent, is a mono-functional/bi-functional agent containing a chloroethylating group11, acting through a mechanism largely based on DNA interstrand cross-linking .The multivariable analysis investigated whether patient- (gender) or treatment-  specific risk factors could be predictive of the FTM clinical benefit. Results of the multivariable analysis demonstrated that time after radiotherapy and second surgery are predictive of a DC in patients treated with FTM. In Figure In the light of these results, we could hypothesize that the conflicting data in the FTM trials could be due to the inclusion of higher risk patients. The GICNO study  also incRecently the EORTC Brain Tumour Group analysed retrospectively data from 300 patients with recurrent GBM, from phase I or II trials, to evaluate patient’s age, sex, World Health Organisation (WHO) performance status (PS), presence of neurological deficits, disease history, use of steroids or anti-epileptics and disease characteristics to predict PFS and OS. This study confirms performance status but not age as a major prognostic factor for PFS and OS in recurrent GBM patients initially treated by chemoradiation with temozolomide . Future Our study is biased in that the analysis was carried out retrospectively, and that the MGMT status data for the patients experiencing tumor recurrence is missing; however, we know that the methylation status of the promoter is prognostic at the time of diagnosis , but doeFTM is confirmed as a valuable therapeutic option for patients with recurrent GBM (PFS-6 m = 33.1%), and is active in all study patient groups . In our study population gender and number of TMZ cycles are not predictive of disease control, while time after the end of radiotherapy and second surgery are independent treatment-related risk factors. These data could be useful to plan prospective and randomized studies to better define the role of chemotherapy in the combination treatment strategy of an heterogeneous cancer, like GBM.TP is an employee of Italfarmaco S.p.A. The study has been supported by an unrestricted grant by Italfarmaco S.p.A. All other authors have no financial or other conflict to declare.AP, IL and SS contributed equally to this work. EB planned and reviewed the statistical analysis. All authors read and approved the final manuscript.


All consumed a control diet for one week, then were randomized to control or DASH for another two weeks (week one and two). With DASH, but not controls, SBP fell by 10.65 ± 12.89 (P = 0.023) and 9.60 ± 11.23 (P = 0.039) mmHg and DBP by 5.95 ± 8.01 (P = 0.069) and 8.60 ± 9.13 mmHg (P = 0.011) at the end of week one and two, respectively. Univariate regressions showed that changes in urinary sodium/potassium ratio (β = 1.99) and plasma renin activity (β = −15.78) and percent change in plasma nitrite after hyperemia were associated with SBP changes at week one . Plasma nitrite following hyperemia showed a treatment effect (P = 0.014) and increased at week two (P = 0.001). Pulse wave velocity decreased over time with DASH (trend P = 0.019), and reached significance at week two (P = 0.026). This response may be mediated by an improvement in upregulation of nitric oxide bioavailability. Early natriuresis and reductions in oxidative stress cannot be ruled out. Future studies are needed to verify these findings, assess the possibility of earlier effects, and examine other potential mediators.Potential blood pressure- (BP-) lowering mechanisms of the DASH dietary pattern were measured in 20 unmedicated hypertensive adults in a controlled feeding study. At screening, participants averaged 44.3 ± 7.8 years, BMI 33.9 ± 6.6 Kg/m Randomized, controlled feeding trials have established the blood pressure- (BP-) lowering effects of the Dietary Approaches to Stop Hypertension (DASH) dietary pattern , 2, whicTherefore, the overall goal of this pilot project was to determine the mechanism(s) by which the DASH dietary pattern lowers BP in unmedicated hypertensive individuals.This study was a randomized controlled feeding trial testing the effects of two dietary patterns on sodium excretion, plasma renin activity, aldosterone, catecholamines, and markers of vascular endothelial function. The design and flow of the study is illustrated in 2, not pregnant or nursing, and willing to be randomized to either of the treatment groups. Exclusion criteria included secondary hypertension, diabetes requiring insulin or hypoglycemic agents, special dietary requirements incompatible with study diets, comorbid medical condition such as active cancer, heart or lung disease, kidney disease (estimated GFR < 60 mL/min), or other major illness, consuming more than 14 alcoholic drinks per week, or major psychiatric disorder. All potential participants completed a phone pre-screening interview first to establish major eligibility criteria, and then attended two face-to-face screening visits to further establish eligibility and obtain pre-run-in measures.Participants were included in the study if they were age 22 yrs or older, stage 1 hypertensive (average SBP 140–159 and DBP 90–99 mmHg over two screening visits), not taking BP medication for at least 3 months prior to screening, had a BMI within 18.5–40 kg/mThe nutrient profiles of the two diets are presented in This study employed the same controlled feeding protocol and followed the same quality assurance procedures used in the DASH and the DASH-sodium multicenter trials , 2. All At the beginning of the study, energy  requirement for each participant was estimated by using the WHO formula adjusted with a physical activity factor estimated from a seven-day physical activity questionnaire . During Randomization occurred electronically in the middle of the run-in period to allow time for kitchen staff to prepare foods. Participants were not notified of their randomization assignment but were likely to infer it when they received the first meal of the randomized feeding. The inability to fully blind study participants to treatment assignment is a known and unavoidable limitation of most feeding studies.All study measurements were performed by trained, certified study personnel during face-to-face clinic visits. The study personnels who obtained these measurements were blinded to intervention assignments. BP was measured in a standardized manner, using an automated device . At the +), potassium (K+), calcium (Ca++), magnesium (Mg++), phosphorous (P), creatinine, and urea nitrogen excretion. In steady state, urinary K+, P, and urea nitrogen serve as markers of dietary intake of fruits and vegetables, dairy, and protein, respectively. Potential mediators of DASH BP response were measured after an overnight fast (except for 24-hour urine collections) and included the following.Dietary intake prior to the study was assessed by collecting a four-day food record during screening, a commonly used reference method for diet assessment , 11, and+.(i) Natriuresis was assessed by 24-hour urinary excretion of Na(ii) Plasma renin activity (PRA) and aldosterone were assayed in a high-quality commercial clinical lab (LabCorp) from fasting blood samples collected after participants were in sitting position for at least 15 minutes.(iii) Urinary catecholamines (epinephrine and norepinephrine) were analyzed from the 24-hour urinary specimen by HPLC with electrochemical detection in a research laboratory at Duke .(iv) Plasma nitric oxide (NO) metabolite analysis was performed on blood taken from the median-cubital vein in the test arm at rest and 60 sec following an upper arm occlusion for 5 minutes. Briefly, the blood samples were drawn into an untreated syringe and mixed with 1 : 1000 heparin at a ratio of 1 mL blood to 5 uL heparin. The plasma was separated out and frozen in liquid nitrogen immediately following collection and then stored at −80°C until assayed for various NO metabolites by reductive chemiluminescence .(v) Vascular endothelial function was measured by brachial artery flow-mediated dilation (BAFMD). All vascular imaging was performed between 8 am and 11 am, on the left arm, with the participant in a supine position with the forearm extended and slightly supinated. Brachial artery assessments were obtained using high-resolution ultrasound and a 7.5 MHz linear array transducer , at baseline (following 10 min of supine rest), during five minutes of forearm occlusion and continuously on r-wave trigger for 2 minutes following cuff release (hyperemia) as previously described , 15. ArtReproducibility of the BAFMD technique has previously yielded average mean differences in brachial artery diameter change for days, testers, and readers of 1.91%, 1.40%, and 0.21 mm, respectively, with intraclass correlation coefficients of 0.92, 0.94, and 0.90, respectively .(vi) Vascular Stiffness was measured by utilizing radial artery pulse wave analysis (RAPWA) and carotid-posterior tibial pulse wave velocity (PWV). All artery pressure waveforms were measured using a Millar tonometer and processed using dedicated software . The system was used to measure an averaged radial artery waveform, derive a corresponding central aortic pressure waveform , then cat-test if continuous variables or Fisher's exact test if categorical data. Three analytical strategies were subsequently used: First, changes over time between the two treatment groups were examined by repeated measure MANOVA tests and comparison between treatments or visits were further conducted when there is a significant time (visit) or treatment effect. Second, changes over time were examined by trend analysis within each treatment group. Lastly, simple univariate regression was used to explore the relationship between changes in each variable with changes in BP. Variables that showed a significant association in the simple regression were then included in a multivariate model. Data collection was complete for all but one of the twenty participants, who discontinued after the week one assessment. BP and other outcome data from this participant were carried forward from week one to the end of week two. The significance level was set at P < 0.05. All data were presented as mean ± SD.All analyses were performed by using the SAS software . Initially, primary outcome data (SBP and DBP) were tested for normality using quantile-quantile plot. Background characteristics were compared between the two treatment groups by using 2(consistent with class I obesity), BP of 144.2 ± 9.38/88.5 ± 6.03 mmHg (P < 0.05).A total of 20 adult participants with unmedicated stage 1 hypertension completed the study. They averaged 44.3 ± 7.8 yrs of age, had a BMI of 33.9 ± 6.6 Kg/m.03 mmHg . Among tF = 5.46, P = 0.023 and F = 9.73, P = 0.003, resp.). As shown in P = 0.023), with sustained effect at week two . Similarly, DASH lowered DBP to a near-significant degree at week one  as compared to the baseline, with statistically significant reduction at week two . Both SBP and DBP remained constant with exposure to the control dietary pattern. The difference between DASH and control approached statistical significance for SBP at week one only (P = 0.056) and were statistically significant for DBP at both week one and week two .The MANOVA test indicates that there were significant treatment (dietary pattern) effects on both SBP and DBP  Natriuresis. Urinary Nabaseline ).P = 0.004). P = 0.022), but the difference was attenuated at week two (P = 0.071). Similarly, plasma aldosterone tended to increase in the DASH group, but there were no significant differences between the treatment groups.(ii) RAAS. Overall, changes in PRA showed a significant treatment effect (MANOVA (iii) Adrenergic tone. Neither urinary norepinephrine nor epinephrine showed any changes during the study. 2−) level following hyperemia showed a significant treatment effect (P = 0.014) in MANOVA (2−) after hyperemia started to show an increase at week one as compared to the baseline, and the increase was significant at week two (P = 0.001). This increase was greater than that of the controls at week two (P = 0.053). This pattern was also observed in the change of plasma nitrite after hyperemia and that the change in DASH increased significantly at week two (P = 0.053), whereas there was a significant decrease within the controls over time (P = 0.026). Similar pattern was observed in the percent change in plasma nitrite after hyperemia. Neither BAFMD nor AIx changed during the course of the study. PWV decreased over time in the DASH group (trend P = 0.019), and the reduction at week two was significant compared to baseline (P = 0.026). (iv) Vascular measurements. Among all the vascular measurements, plasma nitrite (NOn MANOVA . Plasma + (β = 0.12), K+ (β = −0.19), Na+/K+ ratio (β = 1.99), and PRA (β = −15.80) showed a significant association with changes in SBP at week one . Percent change in plasma nitrite after hyperemia also showed a significant negative association with change in SBP . When the urinary Na+/K+ ratio, PRA, and percent change in plasma nitrite after hyperemia were included in a multivariate regression model, all three variables remained significantly associated with SBP change (P < 0.05). Similar pattern is observed with DBP change (data not shown). In simple linear univariate regressions , changesOur data suggest that DASH may lower BP by increasing NO bioavailability, as measured by plasma nitrite following a stressor, and possibly has subsequent effects on vascular basal tone (reduced PWV). There was no evidence that BP reduction was due to effects of DASH on natriuresis, suppression of the RAAS , or on adrenergic tone (as reflected in changes in epinephrine and norepinephrine). Due to small sample size and other limitations discussed below, these data should be considered preliminary evidence necessitating further study. +, a rich component in DASH [Although our previous research showed that DASH may shift the pressure-natriuresis curve  and that in DASH , 21. Fur in DASH . TherefoOur data do not provide evidence that DASH lowers BP by direct effects on the RAAS. Renin activity, the rate-limiting step in generation of the vasoconstrictor angiotensin II, was actually increased relative to changes in the control group, possibly due to compensation for lower BP. We did not measure other RAAS mediators that might reveal a role of the RAAS; indeed, we know that a polymorphism of the angiotensinogen gene is associated with the BP response to DASH . Therefoβ2-adrenergic receptor gene [Our data also provide no evidence that DASH inhibits the adrenergic nervous system. Neither urinary norepinephrine nor epinephrine changed with DASH, but these are fairly crude measures of adrenergic tone. Changes in adrenergic tone can be episodic, and multiple urine collections may be necessary to reflect the change. Indeed, there is evidence of genetic modulation of the DASH effect by a polymorphism of the tor gene . Thus anThe increases in stimulated plasma nitrite in DASH relative to controls suggests that the DASH dietary pattern may improve the vascular endothelium's ability to up-regulate NO, in response to a localized stressor. The DASH dietary pattern contains nuts which are a good source of L-arginine, a precursor of nitric oxide (NO) , 24. OxiThe possibility that the BP-lowering effect of DASH may be mediated through its impact on the vascular endothelium is further suggested by the observed reduction in PWV. Previous research has shown that PWV is an independent predictor of increasing SBP over time and of incident hypertension among people with normotension or untreated hypertension, respectively . Our stu2+ data may suggest another possible BP-lowering mechanism. As noted above, urinary Ca2+ remained unchanged in both treatment groups despite a much higher intake in the DASH group than in the controls. Our previous study showed that the combined effects of higher potassium and low acid load of the DASH diet may contribute to Ca retention and subsequent reduced bone turnover [The urinary Caturnover . Thus coThis study is limited primarily by the small sample size. A controlled feeding protocol, which was used in, and is one of the strengths of, this study allows for near-absolute control over nutrient intake in humans, eliminating extensive confounding that occurs in studies in which participants select and prepare their own meals. However, feeding studies are limited by the resources required and participant burden. Nonetheless, large feeding studies with adequate power are feasible , 2, 36. This study sheds light on the possible mechanism(s) through which the DASH dietary pattern lowers BP. Confirmation in larger populations, evaluation of other potential mediators, and a deeper understanding of molecular mechanisms and pathways are needed. Understanding the BP-lowering mechanisms of DASH can lead to more refined nutritional recommendations, the identification of new drug targets, greater insight into normal and abnormal BP regulation, and more targeted treatments for individual patients.


At the age of 3-4 years, body composition was assessed by dual-emission X-ray absorptiometry followed by postmortem examination. Compared with N animals, male, but not female, offspring of all UN groups had greater % fat mass  and perirenal fat , and proportionately smaller hearts (4.5 ± 0.1 versus 5.2 ± 0.2 g·kg−1), lungs (9.1 ± 0.2 versus 10.6 ± 0.5 g·kg−1), and adrenals (0.06 ± 0.002 versus 0.08 ± 0.003 g·kg−1). UN males also had larger testes , but UN females had smaller ovaries . Changes were independent of birth weight or postnatal growth velocity. Brief PCUN has sex-specific effects on adult phenotype, predominantly affecting males, which may contribute to adverse metabolic outcomes.Periconceptional undernutrition (PCUN) in sheep alters fetal growth and metabolism and postnatal growth regulation, but effects on adult body composition are unknown. We investigated the effects of PCUN on adult phenotype. Singleton lambs of ewes fed normally (N,  Maternal undernutrition around the time of conception in sheep alters fetal metabolic  and endoin utero growth restriction, occurring because of a mismatch of nutrient availability before and after birth, increases relative fat mass thereby conferring an increased risk of later metabolic disease [in utero growth restriction [A number of studies suggest that rapid postnatal growth particularly after  disease –11. Howetriction . Furthertriction . in utero and postnatal growth and development to later metabolic disease risk. The aim of this study was to determine the independent effects of timing and duration of maternal undernutrition; growth during different time periods; offspring sex on later phenotype. We hypothesised that periconceptional undernutrition would result in sex-specific changes in body composition in adulthood, and that these effects would be mediated through altered postnatal growth patterns. We measured growth and body composition from birth to midadulthood in male and female singleton offspring of ewes undernourished before, both before and after, or only after conception, and carcass and organ weights at postmortem. Longitudinal experimental animal studies provide one way of determining the relative contributions of periconceptional nutritional insult, and Ethical approval for the study was obtained from the University of Auckland Animal Ethics Committee. Five-year old Romney ewes were randomly allocated to a control group (N) or to one of three periconceptionally undernourished groups. All ewes received the same high-quality complete feed  . NormallLambs were weighed and measured weekly to 12-weeks (weaning) and then intermittently to 3-4 years of age. Growth velocity was calculated using an exponential method .−1 and ketamine 100 mg·mL−1 intravenously. Fat and lean mass were calculated using Norland software in an area defined by the thoracic inlet proximally and the base of the tail distally, and from the animal's back superiorly to the base of the humerus and femur inferiorly, and expressed as a percentage of body weight. At 3-4 years of age, body composition was assessed by dual-emission X-ray absorptiometry  . Prior tPostmortem examinations were carried out shortly after DXA scanning (3-4 years of age), after pentobarbitone euthanasia. Body measurements included live and eviscerated weight, measurements of linear growth , and chest and abdominal circumferences. Organs were removed quickly after euthanasia and weighed. Data were analysed using StatView (“StatView for windows” SAS Institute Inc. v 5). Groups and sexes were compared using ANOVA, and relationships between growth parameters  and body composition , assessed using regression.Postmortem examinations were performed on 76 animals between 3 and 4 years of age (s of age . P = 0.02), but growth velocity to weaning (GV0-12) was greatest in the N group, so that by 12 weeks, weight was not different among groups. In females, there was no effect of nutritional group on birth weight or postnatal growth velocity. At the time of DXA scanning and postmortem examination, there was no difference among nutritional groups in live weight of animals of either sex. Males were heavier overall than females at birth and in adulthood . In maleP < 0.0001 for each comparison). Males in all UN groups had greater % fat mass  and tended to have lesser % lean mass (P = 0.06) than male controls (N) (postmortem (P = 0.008). In females, there was no difference among groups in adult body composition.Adult females in all groups had greater % fat mass and lesser % lean mass than adult males (rols (N) . They alpostmortem examination, males in all UN groups had decreased shoulder height compared with N males (P = 0.04); males in UN-61-0 and UN-61-30 groups also had shorter body length (P = 0.04). Hind limb length tended to be greater in controls (P = 0.09), with the difference only significant between N and UN-61-0 groups (P = 0.03). There were no differences among the different UN groups, or between UN and N groups, in abdominal or chest circumference. In females, there were no differences among groups in body proportions.At Adult % fat mass was not related to birth weight in males or females of any group. R2 = 0.13, P = 0.04), with the effect strongest in the N group . The association between % fat mass and body weight was even stronger at the time of postmortem , so that the heavier female sheep in all nutritional groups were also fatter. In males, there was no relationship between adult % fat mass and body weight at 12 weeks or at the time of postmortem. However, in females, adult % fat mass was positively associated with body weight at 12 weeks  in males or females from any group. R2 = 0.1, P = 0.09; UN-61-30 males R2 = 0.56, P = 0.008; all females R2 = 0.25, P = 0.002). Adult % lean mass was not related to birth weight, 12 week weight, or early growth velocity in males or females of any group, but in both sexes it was inversely associated with body weight at postmortem, with the strongest relationship in females and UN-61-30 males .Periconceptional undernutrition affected total gonad weight in animals of both sexes, but in opposite ways, with UN males having heavier testes and UN females lighter ovaries than N animals  and twin conception. One study of children born after IVF found perturbed body fat distribution in childhood compared with spontaneously conceived controls [in utero nutritional insult, it may not be causal; rather, it may be another manifestation of the developmental perturbation resulting from periconceptional undernutrition. This hypothesis is further supported by the findings of epigenetic changes after periconceptional undernutrition in fetal hypothalamic genes involved in feeding regulatory networks, including proopiomelanocortin (POMC) and glucocorticoid receptor (GR) [Increased fat mass despite normal body mass index (BMI), termed “normal weight obesity” (NWO), has been increasingly recognised in humans as a risk factor for cardiovascular disease, dyslipidaemia, and the metabolic syndrome , 22, witcontrols , althougcontrols . A studycontrols , again stor (GR) .It has been suggested that rapid postnatal growth may be the pathway by which body composition is altered, and the risk of metabolic disease thereby increased after fetal growth restriction . HoweverAlthough duration and severity of undernutrition in animal studies vary considerably in the published literature, sex specificity of metabolic outcomes is a common finding. Smith et al. found increased insulin secretion in response to a glucose load at 10 weeks of age in male offspring of ewes actively losing weight at the time of conception , whereasOne cannot assume a direct relationship between phenotype and organ function. Adrenal size and function have been investigated in a number of species under different stressors, but the relationship between them, particularly in a basal state, is unclear and may differ between species and under different conditions , 31. We in utero environment leads to prioritising of investment into early growth and reproductive capacity, despite later “trade offs” in size and function of other organs [in utero, rather than by later weight gain, with the onset of puberty at a lower body weight and lower plasma preoestrus progesterone concentrations after maternal undernutrition [The gonadal findings are interesting, and particularly the opposite effects of maternal undernutrition on gonad size in males and females. It has been suggested that an adverse r organs , but malr organs , 34. Femr organs . In femautrition . Howeverutrition . We haveIn utero testicular development is dependent on circulating fetal gonadotrophin concentrations, but the effect of the nutritional environment before fetal pituitary function is established is less clear. Maternal undernutrition for 50 days after mating in sheep did not affect testicular size, but was associated with increased testosterone production, advanced development of seminiferous tubules, and increased expression of steroidogenic acute regulatory protein (StAR) at day 50, implying that undernutrition may upregulate fetal testicular steroidogenesis [In men, testicular dimensions and volume are correlated with testicular function , 39. In ogenesis . We did While serial assessments of body composition throughout the life course would have been desirable, this was not possible due to logistic constraints. The effect of ageing on fat mass might be more profound in males, and there may have been changes over time in body composition that were related to other growth parameters, for example, rapid seasonal weight changes or growth velocity in different time periods, or the timing of puberty. The attrition over time associated with large cohorts of animals over years also meant that numbers in the male control group were lower than ideal. Despite this, the magnitude of the phenotypic differences between controls and undernourished male offspring suggests a profound effect.Brief undernutrition around the time of conception in sheep alters adult phenotype in male offspring, including an increase in proportional body fat. This is likely to increase the risk of metabolic dysfunction and may contribute to the observed effects of periconceptional undernutrition on endocrine and metabolic regulation. The increased gonadal size in males, despite decreased growth of several other organs, may reflect prioritising reproductive capacity in response to an adverse periconceptional environment.


Background. The serum levels of neopterin, a marker associated with cell-mediated immunity are elevated in chronic kidney disease (CKD). We evaluated serum neopterin levels and investigated its association with markers of inflammation in a cross-section of CKD subjects without known cardiovascular disease. Methods. Serum neopterin levels were measured in 118 patients with stage 3–5 CKD and 41 healthy subjects with normal kidney function (HC). Patients with known cardiovascular disease were excluded. We also estimated highly sensitive CRP (hsCRP) and interluekin-6 (IL-6), tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ) in the CKD subjects. All assays were done using commercially available ELISA kits. The correlation between neopterin and markers of inflammation were investigated. Results. Of the CKD population, 82 were in stage 5 (60 stage 5 D), 24 in stage 4, and 12 in stage 3. The mean age was 51.04 ± 1.3 years and 66% were males. The commonest cause of CKD was diabetes (36%). Serum neopterin levels were 5-fold higher in CKD patients as compared to HC . There was a graded increase of serum neopterin from stages 3 to 4 and 5. CKD 5 D patients exhibited significantly higher levels compared to nondialysis stage 5 patients (P < 0.0001). An inverse correlation was noted between serum neopterin and eGFR . Serum neopterin correlated with hsCRP , IL-6 , and IFN-γ  but not with TNF-α. Conclusion. Serum neopterin level is elevated and correlates with the severity of CKD. The elevation correlates with elevation of most, but not all, inflammatory markers. Its role in future development of cardiovascular disease and modulation with anti-inflammatory therapies needs further studies. Neopterin levels are elevated in conditions of T-cell or macrophages activation [Neopterin, a pyrazino-pyrimidine compound, is synthesized by monocytes and macrophages in response to interferon-(IFN-) tivation . It enhativation . Neopterin is a marker of cellular immune response . Its levRecent studies have shown a strong association of neopterin with CVD , 16. Sasα have been demonstrated to be associated with increased cardiovascular mortality in dialysis patients [Chronic kidney disease (CKD) subjects are at a high risk of developing CVD. The CVD risk goes up with declining GFR and dialysis patients have a 35-fold greater CVD mortality risk . High prpatients , 25. ThiSerum and urine neopterin levels are elevated in patients with kidney diseases –28. The Over a 6-month period (July–December 2011), 118 patients with stage 3–5 CKD were recruited in this cross-sectional study from the outpatient clinic and ward population of nephrology department, Postgraduate Institute of Medical Education and Research, Chandigarh, a large multispecialty hospital in north India. Patients over the age of 70, those with known coronary artery disease, heart failure, malignancies, active infections and taking drugs that can modulate inflammatory response were excluded. Out of these, 58 subjects were on hemodialysis (HD) and 60 were stage 3–5 predialysis CKD subjects. Estimated glomerular filtration rate (eGFR) was calculated using MDRD formula , and CKDα, IFN-γ , hsCRP , and neopterin . All the samples were analyzed in duplicate. These markers were selected because of their documented association with CV complication in CKD and their association with serum neopterin level in other diseases.After recruitment, subjects were asked to report in a fasting state. Blood samples were collected in a vacutainer, serum was separated and stored in cryovials at −80°C until analysis. Commercially available enzyme-linked immunosorbent assays kits were used to measure serum IL-6 and TNF-U test, independent t test, and Pearson and Spearman correlation tests were performed as appropriate. All P values were calculated two-sided, and a value of <0.05 was considered significant.Data were expressed as mean ± standard error of mean (SEM) when distributed normally, or as median (interquartile range). Statistical Package for Social Sciences (SPSS) v 16.0 was used to analyze the data. Mann-Whitney Detailed characteristics of study subjects are shown in P < 0.0001). There was no effect of diabetes, hypertension, smoking, age, or gender on the levels. A graded increase was noted in neopterin level from CKD stage 3 to 5 , and had positive associations with hsCRP , IL-6 , and IFN-γ  in the CKD subjects  and IFN-γ  were also significantly higher in dialysis group. The IL-6 level was also increased in dialysis patients but difference was not significant . No difference was noted in TNF-α level.We further subdivided stage 5 CKD patients to dialysis and nondialysis groups. Neopterin level was significantly higher in dialysis group as compared to non-dialysis group . We did not note any correlation between serum neopterin and TNF-α levels. Neopterin levels show continuous rise with falling GFR, and are further increased in dialysis patients. The finding of inverse association of renal function with serum neopterin and a stepwise increase in serum neopterin with increasing stages of CKD is consistent with previous reports [The present study shows that CKD patients exhibit a significant increase in serum neopterin levels compared to healthy subjects, and this increase is correlated with increased circulating levels of several markers of inflammation (hsCRP, IL-6, and IFN- reports , 33 and γ, and TNF-α values in the CKD subjects are significantly higher than those previously reported by us in non-CKD subjects [CKD subjects showed evidence of inflammatory activation at all stages. The CRP, IL-6, IFN-subjects , 30, 34.subjects , IL-18 [subjects , leukocysubjects , fibrinosubjects , hyalurosubjects , CRP [40subjects . IL-6 issubjects . C-reactsubjects . Moreovesubjects –23. Atteγ in CKD suggests a link with the overall state of inflammation. Our study also demonstrated a twofold increase in neopterin levels in dialysis patients as compared to those not on dialysis, but with comparable GFR. This suggests that dialysis might further increase the production of neopterin. These subjects also showed higher levels of inflammatory markers, probably as a result of inflammatory activation and cytokine generation by activated leukocytes as a result of interaction between blood and dialysis membranes [The positive association of serum neopterin level with hsCRP, IL-6, and IFN-embranes  which thThe finding of increased neopterin levels in CKD could have clinical significance. Neopterin can predict morbidity and mortality in chronic inflammatory and infectious diseases . Serum nOur study had some limitations. This is a cross-sectional study, and measurements have been done at a single time point. Most of our patients had newly diagnosed CKD. Whether the relationship still holds in patients on long-term dialysis is not certain. Also, whether these findings are valid for patients from other geographic areas and/or ethnic backgrounds needs to be confirmed. Our patients did not have overt CV disease but still showed rise in neopterin levels. It would be interesting to follow up these patients to see if increased levels predict future development of vascular disease. If this association is confirmed, a role could be suggested for therapies that could modulate inflammatory activation and/or target neopterin.In summary, CKD patients exhibit elevated levels of serum monocyte activation marker neopterin. The degree of elevation correlates with the stage of kidney disease and inflammatory activation. This association needs to be confirmed in larger studies, and the link between inflammatory activation and elevation in serum neopterin levels and their contribution to the clinical events, especially CVD, requires further studies.