Seasonality of ovulatory activity is observed in European sheep and goat breeds, whereas tropical breeds show almost continuous ovulatory activity. It is not known if these tropical breeds are sensitive or not to temperate photoperiod. This study was therefore designed to determine whether tropical Creole goats and Black-Belly ewes are sensitive to temperate photoperiod. Two groups of adult females in each species, either progeny or directly born from imported embryos, were used and maintained in light-proof rooms under simulated temperate (8 to 16 h of light per day) or tropical (11 – 13 h) photoperiods. Ovulatory activity was determined by blood progesterone assays for more than two years. The experiment lasted 33 months in goats and 25 months in ewes.Marked seasonality of ovulatory activity appeared in the temperate group of Creole female goats. The percentage of female goats experiencing at least one ovulation per month dramatically decreased from May to September for the three years . Tropical female goats demonstrated much less seasonality, as the percentage of goats experiencing at least one ovulation per month never went below 56%. These differences were significant.Both groups of temperate and tropical Black-Belly ewes experienced a marked seasonality in their ovulatory activity, with only a slightly significant difference between groups. The percentage of ewes experiencing at least one ovulation per month dropped dramatically in April and rose again in August  or September (temperate ewes). The percentage of ewes experiencing at least one ovulation per month never went below 8% and 17%  during the spring and summer months.An important seasonality in ovulatory activity of tropical Creole goats was observed when females were exposed to a simulated temperate photoperiod. An unexpected finding was that Black-Belly ewes and, to a lesser extent, Creole goats exposed to a simulated tropical photoperiod also showed seasonality in their ovulatory activity. Such results indicate that both species are capable of showing seasonality under the photoperiodic changes of the temperate zone even though they do not originate from these regions. Seasonality of reproduction is a common feature in sheep and goat breeds of temperate latitudes ,2 and seWhen transferred to equatorial conditions , Suffolk ewes (a European breed) cycled at irregular intervals with no clear anovulatory season . In contLocal breeds of sheep -15 and gIn the present experiment, seasonal ovulatory activities were assessed in tropical Creole goats and Black-Belly ewes. These two breeds originate from the Carribean Islands, where they have been bred for 3–4 centuries, and constitute the progeny of African tropical breeds (see Methods). The animals of the present experiment were imported into Europe and experimentally subjected for more than two years in light-proof rooms to an annual photoperiodic regimen simulating that of temperate regions (TE group), and compared to females under tropical cycle simulating that of a tropical region (TR group).Ovulatory activity demonstrated marked differences between experimental groups over the course of the experiment. Individual ovulatory activity is presented in Figure Marked seasonality of ovulatory activity appeared in female goats of the TE group; the seasonal inactivity occurred from May to September for the three years of the study Figures  &2. The In contrast, female goats exposed to the TR photoperiodic cycle demonstrated much less seasonality as the percentage of goats experiencing at least one ovulation per month never went below 56% . Two female goats cycled continuously during the course of the experiment, one female goat in mid-1990 and three in mid-1991 continued their ovulatory activity during spring and summer, and four females were still cycling at the end of the experiment (June 1992).nd and 3rd breeding seasons were significantly higher in TR goats  Experimental ewes were artificially raised in TE vs TR photoperiod from birth to 6 months old from the start of the experiment, whereas female goats were raised under simulated tropical photoperiod until the start of the experiment at one or two years old. (c) Black-Belly ewes were included in the experiment at 6 months of age, whereas Creole goats were 12 and 24 months old at the start of the experiment. These three main differences between the experiments in female goats and ewes could possibly explain this photoperiod x species interaction. However, it may also come from a real difference of sensitivity to non-photoperiodic factors  between the two species.In contrast, Black-belly ewes maintained under simulated tropical photoperiodic variations did not differ greatly from those maintained under simulated temperate photoperiod. The two groups of ewes and goats used here differed in various parameters, some of which could explain the photoperiod x species interaction observed here: (a) Recipient ewes in which embryos were implanted in autumn were maintained under natural photoperiod. It is known that light changes during pregnancy may affect the progeny's photoperiod sensitivity, especially regarding the onset of puberty in sheep -25 and iThe spontaneous ovulatory activity demonstrated in the present experiment by the Black-Belly ewes from the TR group were very different from those registered earlier in their natural breeding conditions on the island of Martinique in the tropics, where they cycled all year round . The CreIn Syrian hamsters, exercise by access to a running wheel can completely inhibit the short-day induced regression of the testis ,32. ExpeThe sheep and goat breeds used here are local breeds of the Carribean islands. Even though their presence in these islands is associated with human history, they are considered as the progeny of tropical but not European ancestors, because they originate from the West coast of tropical Africa (see Methods). In these areas all sheep and goat breeds are considered as non or low seasonal breeders. Thus, the sensitivity of the Creole goats and Black-Belly ewes observed here under temperate photoperiod could be hypothetized as a true sensitivity of these breeds, not a simple inheritance of a trait coming from European ancestors.A marked seasonality in the ovulatory activity of tropical Creole goats and Black-Belly ewes was induced when females were exposed to a simulated temperate photoperiod. Unexpectedly, and differing from the results obtained in their original breeding location, Black-Belly ewes and, to a lesser extent, Creole goats exposed to a simulated tropical photoperiod also showed significant seasonality in their ovulatory activity. Such results indicate that both species are capable of showing seasonality under the the photoperiodic changes of the temperate zone even though they do not originate from these regions.Experimental animals from both species were produced after importation of deep-frozen embryos. The embryos were thawed and re-implanted into recipient females of Saanen goats for Creole embryos (1983) and of Ile-de-France sheep for Black-Belly embryos (1997).th-18th centuries [rd and 4th generations, constituting a sufficient number of animals, made up the two experimental groups.Embryos from donor Creole goats were collected as previously described  from 4 genturies -37. Creoenturies ,37. Commenturies , which rEmbryos from donor Black-Belly ewes were collected using the technique described by Heyman et al. . The donBoth experiments were performed at the INRA Station near Tours (latitude 47°25 North).- Thirty three Creole female goats were used from October 1989 when the animals were one  and two  years old, for 33 months to June 1992 . They were divided into two groups (n = 17 TR and 16 TE) in visual and tactile contact, with entire and vasectomized Creole bucks, but separated by a fence. Each group was maintained in a separate light-proof room throughout the experiment.Feeding conditions were constant throughout the experiment. The animals were fed once daily with a diet of 240 g of barley, 60 g of wheat, 700 g of meadow hay and 300 g of barley straw. No flushing was performed. They had free access to water and to mineral blocks containing oligoelements and vitamins.- Twenty four Black Belly ewes were used from September 1998 when the animals were 6 months old, for 25 months, to October 2000. They were divided into two groups (n= 12 TR and 12 TE) in visual and tactile contact, with entire Black Belly rams, but separated by a fence. Each group was maintained in a separate light-proof room throughout the experiment.Feeding conditions were constant throughout the experiment. The animals were fed once daily with a diet of 150 g of corn, 110 g of barley, 45 g of dehydrated protein complement and 400 g of hay. No flushing was performed. They had free access to water and to mineral blocks containing oligoelements and vitamins.Within each species, one group was subjected to the large photoperiodic changes prevailing at the 45° North latitude (8 to 16 h of light per day from winter to summer solstices); this group was called the temperate group (TE). The other group was subjected to the limited photoperiodic changes prevailing at the 16° North latitude (11 to 13 h of light per day from winter to summer solstices); this group was called the tropical group (TR). In all rooms, photoperiod was regulated by an electric clock that operated bulbs providing an intensity of 300 lux, lateral to the animals' eyes. The photoperiod was adjusted by 15 min shifts  to produce a complete photoperiodic cycle every 365 days.2). Temperature was not controlled but variations were parallel to those monitored outside but with a lower amplitude .The four rooms were adjacent and of the same size  kg in group TE and 25.5 (± 3.0) kg in group TR. Liveweight was measured monthly and showed a regular increase until the end of the experiment (55.7 ± 7.5 and 56.2 ± 5.2 kg for TE and TR respectively). Liveweight of Black-Belly at the beginning of the experiment was 33.2 (± 2.5 sd) kg in group TE and 31.9 (± 2.7) kg in group TR. Liveweight was measured monthly and showed a regular increase until the end of the experiment (49.5 ± 5.9 and 47.0 ± 5.8 kg for TE and TR respectively).Ovulatory activity was assessed twice weekly in goats up to end October 1991 and once weekly thereafter; and once weekly in ewes, using blood samples for the progesterone radioimmunoassay. A rapid assay was performed using the technique described by Terqui and Thimonier . When prThe first occurrence of a positive Progesterone sample was considered as the date of the first ovulation of the season, and the last occurrence of a positive Progesterone sample was considered as the date of the last ovulation of the season. Mean duration of ovulatory activity is the number of days between first and last ovulation in the same breeding season. Mean duration of anovulation is the number of days between last ovulation in a breeding season and first ovulation of the next season. Females which did not present cessations of their ovulatory activity were not included in the calculations of duration of the breeding seasons or duration of the anovulatory periods. In September and October 2000, individual blood samplings were stopped in ewes that had resumed their ovulatory activity.2 method. .Mean dates of onset and end of the breeding season, durations of the breeding season and of anovulatory activity were compared between groups using an unpaired t-test. Variances were compared with F-Tests. Percentages of females showing at least one ovulation per month were analyzed using the ChiAll procedures were performed in accordance with French legal requirements and with the Ministry of Agriculture authorization for animal experimentation nb A37801 .The authors contributed equally to this work. PC conceived the study, and was responsible for its design and coordination. AD followed the experiment in goats and DC the experiment in sheep. YC and GA performed all the embryo transfer procedures, and were in charge of pathological analyses. AD, DC and PC analysed the data. PC drafted the manuscript. All authors read, corrected and approved the final manuscript.


Healthy life expectancy – sometimes called health-adjusted life expectancy (HALE) – is a form of health expectancy indicator that extends measures of life expectancy to account for the distribution of health states in the population. The World Health Organization reports on healthy life expectancy for 192 WHO Member States. This paper describes variation in average levels of population health across these countries and by sex for the year 2002.Mortality was analysed for 192 countries and disability from 135 causes assessed for 17 regions of the world. Health surveys in 61 countries were analyzed using new methods to improve the comparability of self-report data.Healthy life expectancy at birth ranged from 40 years for males in Africa to over 70 years for females in developed countries in 2002. The equivalent "lost" healthy years ranged from 15% of total life expectancy at birth in Africa to 8–9% in developed countries.People living in poor countries not only face lower life expectancies than those in richer countries but also live a higher proportion of their lives in poor health. World Health Report 2000, the World Health Organization (WHO) reported for the first time on the average levels of population health for its 191 member countries using a summary measure that combines information on mortality and morbidity [In the orbidity .HALE has been calculated previously for Canada and Australia using population survey data on disability -6. The UIt has long been known that health expectancy estimates based on self-reported health status information are not comparable across countries due to differences in survey instruments and cultural differences in reporting of health ,10. The World Health Report 2004 [Given these problems, WHO undertook a Multi-Country Survey Study on Health and Responsiveness (MCSS) in 2000 and 2001 in collaboration with Member States using a standardized health status survey instrument together with new statistical methods for adjusting biases in self-reported health -19. Thesort 2004 . This paCalculation of HALE requires three inputs: life tables, prevalences of various health states, and valuations of time spent in these health states compared to full health. The WHO methods used to calculate HALE have been developed to maximise its comparability across populations. These methods are described in more detail elsewhere ,23 and hProcedures used to estimate the 2002 life tables differed for Member States depending on the data availability to assess child and adult mortality. Complete or incomplete vital registration data together with sample registration systems cover 72% of global mortality. Survey data and indirect demographic techniques provide information on levels of child and adult mortality for the remaining 28% of estimated global mortality. Separate estimates were used for the numbers and distributions of deaths due to HIV/AIDS in countries with a substantial HIV epidemic . A full Following an initial scientific review , signifiBecause comparable health state prevalence data are not yet available for all countries, two sources of information were used: the Global Burden of Disease (GBD) study and the MCSS. Data from the GBD study were used to estimate severity-adjusted prevalences by age and sex for all 192 countries . SecondlThe GBD revisions draw on a wide range of data sources to develop internally consistent estimates of incidence, prevalence, duration and years lived with disability (YLD), for 135 major causes, for 14 sub-regions of the world . PrevaleWorld Health Report 2004 [.Tables Summation of prevalence YLD across all causes would result in overestimation of the total average severity-weighted health state prevalence because of comorbidity between conditions. In earlier calculations of HALE, adjustments were made for independent comorbidity assuming that the probability of having two (comorbid) conditions would equal the product of the probabilities for having each of the diseases. For the World Health Report 2003, further work was undertaken to take dependent comorbidity into account more rigorously . For manData from five large national health surveys were analysed by age and sex to estimate "dependent comorbidity factors" for pairs of conditions. There was surprising consistency in these factors across the five surveys and the results were used for all Member States to adjust for dependent comorbidity in summation of prevalence YLD across all causes .The MCSS was carried out in 2000–2001. A total of 71 surveys were completed in 61 countries using face-to-face, postal and telephone interviewing modes . Thirty-We included in the analysis only those surveys that have met certain explicit criteria that reflect the quality of survey implementation with specific reference to the health vignettes. The USA postal survey was also excluded because respondents were presented the vignettes in order of severity rather than randomized as in the case of all other surveys. Sixty-two surveys met these criteria and were included in the model. Health state prevalences from the WHO Multi-country Household Survey Study were assumed to relate to calendar year 2000. Trends in prevalence YLD between 2000 and 2002 were calculated for each Member State using the GBD estimates. Aggregated across all causes, these trends were generally small. In calculating HALE for 2002, the 2000 survey results were adjusted for likely change over two years using these estimated trends.The health state valuations used in HALE calculations represent average population assessments of the overall health levels associated with different states. They range from 0 representing a state of good or ideal health to 1 representing states equivalent to being dead. These weights do not measure average levels of well-being or quality of life associated with health states, or imply any societal value of a person in a disability or health state. Rather they characterize health decrements on a continuum starting from the societal ideal of good health.Household surveys including a valuation module were conducted in fourteen countries: China, Colombia, Egypt, Georgia, India, Iran, Lebanon, Indonesia, Mexico, Nigeria, Singapore, Slovakia, Syria and Turkey. Data on nearly 500,000 health state valuations from over 46,000 respondents were used to develop a mapping function that captured the average relationships between levels on the six core health domains and overall health state valuations. This average global valuation function was then applied to the vignette-adjusted health domain levels for each survey respondent in order to estimate health state valuations for the calculation of HALE ,31.The MCSS survey samples did not include older people resident in nursing homes or other health institutions. Because these people will generally have worse health than those resident in households, adjustments were made to account for the older population who were resident in health institutions. Fifty-four national estimates of the proportion of the population aged 65 years and over who are resident in nursing homes were collected for 36 countries from national statistical publications and international statistical databases of OECD and the World Bank. These were used to estimate the percentage of the population aged 60+ years institutionalized in MCSS countries. This ranged from 3 to 5% in most OECD countries, was highest at around 7% in the Netherlands and Sweden, was substantially lower at around 0.3 to 1% in Eastern European countries, and was close to zero for developing countries. As data on the severity distribution of health states in institutionalized populations were not available, an average disability weight of 0.5  was assumed for the institutionalized population. Sensitivity analyses showed that the resulting HALE estimates were not sensitive to the choice of this disability weight within a plausible range of variation.Because there is potential measurement error in severity-weighted health state prevalences derived from both household surveys and epidemiological estimates, posterior estimates of prevalence for the survey countries were calculated as weighted averages of the GBD-based prevalences and the survey prevalences . The relHALE was calculated using Sullivan's method  based onApart from ongoing revisions to the GBD analyses of epidemiological information on diseases and injuries, the implementation of improved methods for dealing with comorbidity has resulted in a reduction in estimated proportion of healthy years of life lost at older ages compared to previous years.Improvements in methods used for the analysis of the MCSS survey data and in the adjustment of total YLD rates for dependent comorbidity have resulted in improved estimates of the severity-adjusted prevalence of health states and a reduction in the uncertainty associated with these estimates. For these reasons, HALE estimates for 2002 are not directly comparable with those for 2000 and 2001 published in the World Health Report 2002 .The survey data identified considerable variability in the use of question response categories to describe the same level on a particular health domain and a systematic tendency for people in countries with higher income per capita to use more severe response categories in rating a given anchoring vignette. Thus the self-reported prevalence of problems for a health domain in a country may be quite different from the prevalence of problems after adjustment for these response category cut-point shifts. In France, for example, 70% of survey respondents gave self-report responses of mild or greater problems on the question for the affect domain (anxiety and depression), whereas the standardized (vignette-adjusted) prevalence at all levels higher than "none" was only 33% for France. In contrast, for some countries such as Egypt, the self-report and standardized prevalences were almost the same at around 38%.World Health Report 2004 [Figure Japanese women led the world with an estimated average HALE at birth of 77.7 years in the year 2002, 7.5 years lower than total life expectancy at birth. HALE at birth for Japanese males was 72.3 years, 5.4 years lower than for females. This male-female gap in HALE was narrower than that for total life expectancy at birth (6.9 years). After Japan, in second to seventh places, were San Marino, Sweden, Switzerland, Monaco, Iceland and Italy, with HALE at birth  in the range 72.7 to 73.4 years, followed by Australia and a number of other industrialized countries of Western Europe. There was a considerable range of uncertainty in the ranks for countries other than Japan, with typical 95% uncertainty ranges for HALE of around 0.5 to 2 years for developed countries. Keeping these uncertainty intervals in mind, Canada was in 11th place (72.0 years) and the USA in 29th place (69.3 years).Other countries with reasonably high HALE in the Americas included Argentina (65.3 years), Chile (67.3 years), Costa Rica (67.2 years), Cuba (68.3 years), Mexico (65.4 years), Panama (66.2 years) and Uruguay (66.2 years). Brazil was split, with a high HALE in its southern half, and a lower one in the north. The total average was a relatively low 59.8 years, at 57.2 for males and 62.4 for females.Overall, global HALE at birth in 2002 for males and females combined was 57.7 years, 7.5 years lower than total life expectancy at birth Figure . In otheHALE at birth ranged from a low of 40 years for African males to over 70 years for females in the low mortality regions of Western Europe, North America and the Pacific . This reflects an almost 2-fold difference in HALE between major regional populations of the world Figure . The equThere was a similar almost two-fold variation in HALE at age 60 across the regions of the world, ranging from 19 years for women in low-mortality countries to around 10 years for men and women in sub-Saharan Africa. Males lost more healthy years of life at age 60 than females only in China and South East Asia, where life expectancy gaps at age 60 were also low compared with the more than 5-year gap in Japan  and in Eastern Europe .There was an enormous difference between the world's highest HALE at birth of 77.7 years  and the lowest of 27.2 years  in 2002. In Sierra Leone, people both lived shorter lives , and had higher levels of disease and disability at all ages. The probability of a male child dying before his 5th birthday was 33% in Sierra Leone, compared with less than 0.5% in Japan.The low levels of HALE in sub-Saharan Africa reflect the additional impact of the HIV-AIDS epidemic, as well as war and conflict in some countries such as Sierra Leone. AIDS is now the leading cause of death in Sub-Saharan Africa, far surpassing the traditional deadly diseases of malaria, tuberculosis, pneumonia and diarrhoeal disease. AIDS killed 2.1 million Africans in 2002, versus 300,000 AIDS deaths in 1990 .In Russia, HALE at birth was 64.1 for females, 4 years below the European average, but just 52.8 years for males, 9.4 years below the European average. This was one of the widest sex gaps in the world and reflects the sharp increase in adult male mortality in the 1990s. The most common explanation is the high incidence of male alcohol abuse, which led to high rates of accidents, violence and cardiovascular disease. From 1991 to 1994, the risk of premature death increased by 50% for Russian males . BetweenAt the other extreme there were 12 countries where female HALE at birth was lower than male, and an additional 20 countries where it was less than 1 year higher. These included African countries greatly affected by HIV/AIDS such as Botswana, Kenya, Tanzania and Zimbabwe, but also Eastern Mediterranean countries such as Bahrain, Kuwait, Qatar and the United Arab Emirates, and Asian countries such as Afghanistan, India, Pakistan and Bangladesh. Also included were Nigeria and Haiti. In most of these countries, female life expectancy was slightly higher than male; only in Qatar, Maldives and Bangladesh was female life expectancy at birth lower than that for males. However, higher levels of disability and poor health reduced HALE for females to a greater extent than for males in these countries. At a broader regional level, sub-Saharan Africa, the Eastern Mediterranean region, and the South East Asian region all had female HALE at birth less than three years higher than male, compared with a female-male gap of 6 to 7 years in developed countries.HALE at birth in Afghanistan was estimated at 35.5 years, the 11th lowest in the world in 2002. There was a large 95% uncertainty range around this estimate, of 27 to 44 years, reflecting the lack of population health information for that country. More health information was available for Iraq, where HALE at birth in 2002 was estimated at 50.1 years, with an uncertainty range of 47 to 54 years.China had a healthy life expectancy well above the global average, at 64.1 years, 65.2 years for women and 63.1 for men. Other countries in the Asian region generally had lower HALE. Improving health in Viet Nam has resulted in a HALE of 61.3 years, while Thailand has not improved significantly over the past decade, with a HALE of 60.1 years in 2002. HALE in Myanmar was just 51.7 years at birth, substantially behind its South East Asian neighbors.Figure Correspondingly, LHE varied from 7.3 to 11 years, or by up to 50%. If male and female HALE are considered separately, the range of variation increases to 59–66 at total life expectancy of 72 years. While LHE increases somewhat with increasing life expectancy up to around 70 years life expectancy, there is a flattening for males and a trend to decreasing LHE for females in the countries with longest life expectancies. Although there are higher prevalences of disabling conditions such as dementia and musculoskeletal disorders in countries with longest life expectancies, this is offset by lower levels of disability for diseases such as cardiovascular disease and chronic respiratory diseases where incidence and mortality rates are also lower.As discussed elsewhere, the new methods used in the WHO Multi-Country Survey Study offer clear evidence that self-report data on health status are not comparable, and allow adjustments that improve the comparability of the resulting health status measures . BuildinDespite the fact that people live longer in the richer, more developed countries, and have greater opportunity to acquire non-fatal disabilities in older age, disability has a greater absolute (and relative) impact on healthy life expectancy in poorer countries. Separating life expectancy into equivalent years of good health and years lost to sub-optimal health thus widens rather than narrows the difference in health status between the rich and the poor countries.Richer countries should be much more active in seeking ways to improve the health of the world's poor. WHO has been a strong advocate for efforts to increase the resources available for this purpose, and the recent report of its Commission of Macro-economics and Health concluded that the bulk of the global disease burden is the result of a relatively small set of conditions, each with an existing set of effective interventions . The maiThe World Health Report 2002 included an analysis of mortality and morbidity attributable to the combined effects of 20 selected leading risk factors for 14 subregions of the world ,40. GlobThe regular assessment of levels of population health is a key input to the public policy process, and comparable measurement of population health levels creates possibilities of investigating broad determinants at national and cross-national level. Mortality indicators are not adequate for this purpose, given the considerable policy interest for many populations as to whether – and to what extent – gains in life expectancy have been accompanied by improvements in non-fatal health status ,42-45. TComparability is fundamental to the use of survey results for development of evidence for health policy but has been under-emphasized to date in instrument development. We believe that the new methods used in the WHO Multi-country Household Survey Study and the World Health Survey have increased the comparability of self-report data across countries and provide the first steps towards the consistent and comparable measurement of population health across the world. Final confirmation that compression of morbidity is occuring in low mortality populations awaits longitudinal cross-population comparable data for specific populations: this is now achievable.The author(s) declare that they have no competing interests.CDM, CJLM and JAS developed the methods for calculation of HALE. CJLM, JAS and AT developed the methods and carried out the calculations for the measurement of health states and health state valuations in the MCSS, with contributions from the other authors. CJLM, JAS, SC and BU developed the MCSS survey instrument, and SC and BU coordinated the implementation and analysis of the surveys. KMI, CDM and JAS carried out analyses and calculations for the computation of HALE for the year 2002 for 192 WHO Member States, with contributions from CJLM and the other authors. CDM drafted the paper, with substantial contributions from other authors.The pre-publication history for this paper can be accessed here:


A major goal of cancer research is to identify discrete biomarkers that specifically characterize a given malignancy. These markers are useful in diagnosis, may identify potential targets for drug development, and can aid in evaluating treatment efficacy and predicting patient outcome. Microarray technology has enabled marker discovery from human cells by permitting measurement of steady-state mRNA levels derived from thousands of genes. However many challenging and unresolved issues regarding the acquisition and analysis of microarray data remain, such as accounting for both experimental and biological noise, transcripts whose expression profiles are not normally distributed, guidelines for statistical assessment of false positive/negative rates and comparing data derived from different research groups. This study addresses these issues using Affymetrix HG-U95A and HG-U133 GeneChip data derived from different research groups.We present here a simple non parametric approach coupled with noise filtering to identify sets of genes differentially expressed between the normal and cancer states in oral, breast, lung, prostate and ovarian tumors. An important feature of this study is the ability to integrate data from different laboratories, improving the analytical power of the individual results. One of the most interesting findings is the down regulation of genes involved in tissue differentiation.This study presents the development and application of a noise model that suppresses noise, limits false positives in the results, and allows integration of results from individual studies derived from different research groups. DNA microarrays have become a useful tool in biomedical research as they can be used to determine the relative expression of thousands of genes in a given sample. Such expression profiles could predict genetic predisposition to disease, serve as a set of diagnostic markers, define better drug treatments options for existing diseases (pharmacogenomics), or mark the precise nature of disease progression. A major limitation of this technology is the lack of uniform data mining strategies. This study integrates complementary approaches to more effectively analyze Affymetrix GeneChip microarray data derived from several different types of solid tumors. If the noise is consistent and reproducible it can be filtered from the data and some false positives can be eliminated. There are two principal sources of noise in microarray experiments: biological noise and technical noise. Biological noise consists of variation among patients and tumor locations, variation in the cellular composition of tumors, heterogeneity of the genetic material within tumor due to genomic instability. Technical noise consists of differences in sample preparation and experiment variables which include nonspecific cross hybridization, differences in the efficiency of labeling reactions and production differences between microarrays. Biological noise cannot be corrected but it can be accounted for with statistics using replicates of the treatments or conditions. However, the noise derived from experimental techniques is reproducible and its boundaries can be modeled. It has been observed that in differential gene expression comparisons of any given gene, there is a greater variance in the fold-change calculation at lower signal intensities ,2, and wThe principal concerns when using microarray data derived from different labs to identify cancer markers is that chip-to-chip normalization cannot eliminate differences in cRNA synthesis and labeling, hybridization protocols, scanner settings and image processing software. Variable RNA quality can influence the amount of individual cRNAs generated. The laser power on scanners can differ causing saturation of high intensity probe sets and may have a more variable estimation of the very low expressed transcripts.Two studies ,8 have sThe commonly used t-test tests to see if two populations have a different mean but does not test the overlap of the populations. Selecting markers with minimal overlap in their expression between the normal and tumor states would improve their predictive value. We developed a method to find such markers using an un-weighted voting scheme. This non-parametric method for marker selection was chosen so that no assumptions on the shape of the data distribution were required. The computed noise boundary makes the selection criteria more stringent, eliminating many false positives signals and highlighting genes that are differentially expressed most consistently in comparisons between a cancer and its corresponding normal tissue. This integrative approach can yield a signature of distinct transcripts distinguishing a variety of solid tumors.The objectives of this work were three fold: 1). develop a noise boundary for GeneChip data, 2). develop an algorithm for selecting markers with minimal overlap in their expression between the normal and tumor states, 3). integrate the analysis of previously published data from different sources.The noise boundary model was created to evaluate the reliability of calculated fold changes. Data from normal tissue biopsies obtained from public data sets were used to infer the noise boundary to be used when comparing the normal tissue to their corresponding cancer biopsies. The cancer biopsies were not used in designing the noise model as they are likely to be more variable than normal tissue. Three different signal extraction methods, MAS5 ,10, dChith percentile of the fold-change, chosen as the noise boundary, was calculated for each bin. Figure th percentile error boundaries for the five different tissues as a function of the inverse of the average bin intensity. To decrease the effect of saturation on the regression, the gene expression values in the top 8% were eliminated  of each cancer biopsy to each normal biopsy was determined. Those ratios were recorded and evaluated against the noise boundary model. If a ratio was above the ratio given by the noise boundary, the direction of the fold-change as increased (+) or decreased (-) expression was recorded. If the ratio was below the value given by the noise boundary for the average of the intensities, then the fold-change was considered insignificant and assigned a no change (0) direction.For each probe-seti in the cancer classFor each sample cj in the normal classFor each sample ni>nj then r = ci/nj else r = nj/ciIf ci+nj)/2) And If ci>njIf r>noise_boundary ((cPos_score = Pos_score + 1i+nj)/2) And If ci ≤ njIf r>noise_boundary  and the Er computation was repeated. Each time the data was shuffled, the probe sets were sorted by descending Er score and the probe set information was discarded and replaced by its rank. The average and standard deviation of the ranks was then computed and compared to the results obtained for the cancer versus normal biopsies. For all the comparisons performed, higher Er scores were obtained in the case of cancer versus normal classifications than with randomly shuffled sets. An illustration of the results obtained with the breast cancer versus normal biopsies can be seen in figure To compare the Er algorithm including the noise model to other commonly used analysis methods, the replicate set from the Latin square dataset was used . In thisThe Er model was used to compare each cancer biopsy to its corresponding normal tissue. In the absence of error modeling, the directional change algorithm identified 1,910 probe-sets that had an Er score above 0.9 in ovarian cancer, 1,355 in breast cancer, 1,730 in oral cancer and 322 in prostate cancer. Incorporation of error modeling dramatically reduced the number of probe-sets with Er scores above 0.9 to 272 for ovarian, 177 for breast, 129 for oral cancer and 2 for prostate cancer see . For lunThe advantage of determining Er scores for differentially expressed cancer transcripts is that it provides a statistical metric that can be used to underscore markers that are unique to a particular cancer. Although the Er is not a statistical test and an Er score can vary in its significance depending on the number of samples studied, we selected genes with a high Er index in one cancer type (Er> = 0.9) and low in the others (Er<0.6). As Affymetrix HG-U95A and Hu133A contain different probe-set numbers for the same gene, the SOURCE software  from StaSIM2-s expression in a colon cancer cell line restored growth inhibition and apoptotic cell death [The number of genes found to be reliable markers varied greatly between cancer types [see Additional File ll death .Two genes, AGER and MARCO, were found to be under expressed in all the lung cancer types compared to other cancer types. The advanced glycosylation end product-specific receptor (AGER or RAGE) has been previously reported down-regulated in non small cell lung carcinoma . AGER isThirty nine probe sets were found to have an Er score above 0.9 in ovarian cancer and lower than 0.6 for the other cancers. Two of these genes, Janus kinase 1 (JAK1) and a zinc finger homeobox (ZFHX1B), which are involved in the TGF β signaling pathway regulating cell growth, were down regulated. PAX8, a gene important in development which had been identified in an earlier study , was fouBreast cancer profiles have several interesting features. First, 16 ribosomal protein genes have decreased expression: L34 is involved in translational control , S27 in In oral cancer [see Additional File The method described here provides improved non-parametric approaches to microarray data analysis. After applying the noise boundary model, markers were selected according to their consistency for up-regulation or down-regulation using a voting scheme comparing normal versus cancer biopsies. Tissue-specific expression differences were eliminated by comparing the cancer samples to the normal biopsies from the same tissue. The genes with the greatest differential expression between cancer and normal biopsies were then compared between cancer types. This differs from previous studies ,8,35 whiAll of the microarray data used in this analysis was derived from RNA isolated from biopsies and hybridized on Affymetrix GeneChips HG-U95A, HG-U95Av2 or HG-U133A. All the research groups used the same standard procedure for labeling the cRNA, hybridization and scanning the GeneChips . The datVA and PT conceived the study. VA carried out the analysis and drafted the manuscript. MC performed the programming. JC helped with the evaluation of raw image analysis methods. MR supervised the programming and analysis. PS and JD participated in the study design and management.Assessing the noise level and trust threshold for differential expression on Affymetrix GeneChips. This document compares the noise from MAS5, RMA and dChip and presents the sensitivity analysis for the noise model parameters using the Latin square replicate data set.Click here for fileGene markers for prostate, breast, ovarian, oral, and lung cancer. This file presents the Top Er scores for each cancer studied when compared to its normal tissue. There is a result table with gene information for each cancer on separate tabs.Click here for fileGene markers that distinguish between prostate, breast, ovarian, oral, and lung cancer. This file presents the Er scores of genes expression levels that are consistently up or down in a given cancer compared to its normal tissue (Er>0.9) but not in any of the other four cancers (Er<0.6).Click here for file


Protease inhibitors are known to alter the lipid profiles in subjects treated for HIV/AIDS. However, the magnitude of this effect on plasma lipoproteins and lipids has not been adequately quantified.To estimate the changes in plasma lipoproteins and triglycerides occurring within 12 months of initiating PI-based antiretroviral therapy among HIV/AIDS afflicted subjects.We included all antiretroviral naïve HIV-infected persons treated at St-Paul's Hospital, British Columbia, Canada, who initiated therapy with protease inhibitor antiretroviral (ARV) drugs between August 1996 and January 2002 and who had at least one plasma lipid measurement. Longitudinal associations between medication use and plasma lipids were estimated using mixed effects models that accounted for repeated measures on the same subjects and were adjusted for age, sex, time dependent CD4+ T-cell count, and time dependent cumulative use of non-nucleoside reverse transcriptase inhibitors and adherence. The cumulative number of prescriptions filled for PIs was considered time dependent. We estimated the changes in the 12 months following any initiation of a PI based regimen.A total of 679 eligible subjects were dispensed nucleoside analogues and PI at the initiation of therapy. Over a median 47 months of follow-up (interquartile range (IQR): 29–62), subjects had a median of 3 (IQR: 1–6) blood lipid measurements. Twelve months after treatment initiation of PI use, there was an estimated 20%  increase in total cholesterol and 22% (12% – 33%) increase in triglycerides.Twelve months after treatment initiation with PIs, statistically significant increases in total cholesterol and triglycerides levels were observed in HIV-infected patients under conditions of standard treatment. Our results contribute to the growing body of evidence implicating PIs in the development of blood lipid abnormalities. In conjunction with the predominance or men, high rates of smoking, and aging of the treated HIV-positive population, elevated lipoproteins and triglycerides may mean that patients such as these are at elevated risk for cardiovascular events in the future. Abnormalities in the lipid metabolism of persons infected with human immunodeficiency virus (HIV), potentially induced by the disease itself and the medications used for treatment, were first reported in the early 1990s-5, elevaUp to one-half of subjects treated with PIs have shown elevated levels of triglycerides, total cholesterol (TChol), LDL, insulin, and fasting glucose -10. The We included all antiretroviral naïve HIV-infected persons treated at St-Paul's Hospital, British Columbia (BC), Canada, who initiated use of PIs between August 1996 and January 2002. Plasma lipoprotein and triglyceride levels were obtained from measurements taken over the course of regular monitoring. Longitudinal effects of the impact of PIs on lipoproteins and triglycerides were estimated using statistical models that accounted for correlation due to repeated measurements on the same individuals.In BC, antiretroviral drugs have been centrally distributed at no cost to eligible HIV-infected individuals since 1986,13. In OAt the BC Centre for Excellence in HIV/AIDS, records of CD4+ T-cell counts and a profile of dispensed antiretroviral therapy are routinely maintained, including the: prescription fill dates, medications prescribed, and amount dispensed. Records of dispensed antiretroviral medications,  were used to determine the drugs dispensed and available to each subject during each month of follow-up. Subjects were considered unexposed until the first month that a PI was dispensed. The exposure at the time of the lipid measurement was calculated as the cumulative number of consecutive months of drug exposure up until and including the month in which a measurement was taken. As we were interested in estimating changes that occur in plasma lipoproteins and triglycerides within the first year after treatment initiation, we included measurements with a cumulative PI exposure up to a maximum of twelve months. Subjects were considered unexposed thirty days after the last PI was dispensed and, at that time, the cumulative exposure was set to zero.Approximately one-half of HIV-infected patients in BC are followed at one treatment centre located at St. Paul's Hospital, a large teaching hospital in Vancouver. For these subjects, in addition to virological testing, the hospital laboratory records the results of plasma lipoproteins and triglycerides. Subjects were routinely instructed to fast for 12 hours prior to the sample being drawn. Blood samples were collected in 10-1 EDTA-coated vacutainer tubes. Plasma was separated by centrifugation for 10 minutes at 2,000 revolutions per minute. TChol in the plasma was determined using an enzymatic method and plasFor some subjects, the first lipid measurement was taken prior to the dispensation of any antiretroviral drug ("treatment naïve" baseline lipid measurements). Other subjects had their first recorded plasma lipoproteins and triglycerides after HAART was initiated (initiation lipid levels).© function lme(.). An analysis of variance indicated that simple parallel-line models, where the exposure effect was assumed to be constant over subjects, could adequately explain variation in the data compared to more complex models with random slopes. To account for correlated responses due to repeated measurements on the same individual, the correlation structure of the model was specified to include only a random intercept for each subject.We used linear mixed effects models to estimThe following explanatory variables were included in the models: age, sex, and CD4+ T- cell count at PI initiation, concomitant use of NNRTI, and a measure of adherence to antiretroviral therapy. Exposure to NNRTI was considered time-dependent and quantified using the same algorithm that was used to calculate time dependent PI exposure. Adherence was calculated by dividing the number of months of antiretroviral medications dispensed by the number of months of follow-up in the first twelve months after treatment initiation. Incomplete adherence represents the gap between the time that the previous medication supply ran out until the next refill date, and/or until the last contact date with the program. This method is reliably associated with both clinical outcomes and un-timed drug level monitoring,21.For each outcome and associated regression coefficient β, the quantity exp(β)-1 represented the adjusted monthly percent change in that plasma lipid fraction. These values were annualized using the formula exp(12 × β)-1. Ninety five percent confidence intervals (95% CI) for the effect estimates were obtained using the standard error for each regression coefficient.Two sensitivity analyses were conducted. The first analysis involved restricting the analysis to lipid measurements which were taken on subjects prior to any change from baseline antiretroviral therapy. The second sensitivity analysis included only subjects who had lipid measurements taken prior to initiation of therapy.3. All subjects initiated therapy that included a nucleoside analog and a PI.There were 679 subjects who were eligible for analysis, 91% of whom were male  measurements per subject. The median time between lipid measurements was 3 (IQR 2–5) months for all subjects.Table The sensitivity analyses indicated that the models were robust to changes in the sub-population studied and the exposures that were included. In the analysis that included subjects who had a baseline measurement prior to start of HAART, a plasma profile prior to antiretroviral therapy initiation showed a similar pattern but with wider confidence intervals. In the analysis which was restricted to lipid measurements taken on subjects prior to switching from baseline therapy, the effect estimates were similar to those in Table In patients treated for HIV infection with HAART in a naturalistic setting, we observed that treatment with PIs was estimated to result in 20% increases in the levels of total cholesterol, HDL- and LDL-cholesterol and triglycerides twelve months after treatment initiation. Our results contribute to the growing body of evidence implicating PIs in the development of blood lipid abnormalities and are Low HDL levels and other forms of dyslipidemia, and disturbances in glucose metabolism, as well as central obesity, have been shown to be strong independent risk factors for cardiovascular morbidity and mortality in the general population,26. FurtTwo large observational cohort studies, published in 2003, showed discrepant results regarding the risks of cardiovascular disease among persons treated with HAART,30. A reThis study has a number of features that add credence to the results. First, while only based at one hospital, the study population comprised about one-half of the treated population in BC. The study sample had similar demographic composition (age and sex) and CD4+ T-cell count at initiation of therapy as other treated subjects in the province. We believe that these results are therefore generalizable to PI-regimen treated HIV-infected patients in BC, as well as to other target populations with similar demographic characteristics. Second, as all dispensed PIs and other antiretroviral medications are paid for and recorded centrally, there was complete information on all HAART medications available to all study subjects. We adjusted for adherence using each subjects' refill compliance in the first year of treatment. Third, data on the plasma lipid profile, including both lipoproteins and triglycerides, were of high quality. The measurements, while lacking the feature of being taken at uniformly spaced intervals as in closely monitored system such as randomized trial, represent the actual experience in an observational setting. Fourth, the enrollment period lasted over five years, allowing a large sample size. We employed repeated measures analyses to account for the correlated structure of the data.This study was also subject to several limitations. First, lipid measurements were not collected in a predetermined manner as is typical in a prospective research study. Consequently, the timing of measurements was irregular and not all lipid fractions were measured when blood was drawn. Second, despite instructions some subjects may not have fasted for the full 12 hour period prior to blood draws. Non-fasting triglyceride measurements are still useful for predicting cardiovascular disease and death. Third, Due to changes in the HAART regimen that occurred because of resistance, non-compliance, or other reasons, it was not possible to isolate the effect of individual PIs. Over the period under study, most subjects initiating PI-based HAART were prescribed either indinavir or nelfinavir.As PI-based HAART is very effective in increasing survival in HIV-infected patients, discontStatins have been shown to be effective drugs to prevent cardiovascular disease in non-HIV patients. While tFibrates are effective in lowering triglycerides and increasing HDL and have been shown to be effective in HIV patients. DespiteWe conclude that there was an estimated 20% increase in lipoproteins and triglycerides in the first year after initiating PI-based antiretroviral therapy in HIV-infected patients under conditions of standard treatment. Whether these increases continue beyond one year is of considerable interest because lipoproteins and triglycerides are known risk factors for cardiovascular disease. In conjunction with the predominance or men, high rates of smoking, and aging of the treated HIV-positive population, elevated lipoproteins and triglycerides may mean that patients such as these are at elevated risk for cardiovascular events in the future.


In type 2 diabetes, free fatty acids (FFA) accumulate in microvascular cells, but the phenotypic consequences of FFA accumulation in the microvasculature are incompletely understood. Here we investigated whether saturated FFA induce apoptosis in human microvascular mesangial cells and analyzed the signaling pathways involved.Saturated and unsaturated FFA-albumin complexes were added to cultured human mesangial cells, after which the number of apoptotic cells were quantified and the signal transduction pathways involved were delineated.The saturated FFA palmitate and stearate were apoptotic unlike equivalent concentrations of the unsaturated FFA oleate and linoleate. Palmitate-induced apoptosis was potentiated by etomoxir, an inhibitor of mitochondrial β-oxidation, but was prevented by an activator of AMP-kinase, which increases fatty acid β-oxidation. Palmitate stimulated an intrinsic pathway of pro-apoptotic signaling as evidenced by increased mitochondrial release of cytochrome-c and activation of caspase 9. A caspase 9-selective inhibitor blocked caspase 3 activation but incompletely blocked apoptosis in response to palmitate, suggesting an additional caspase 9-independent pathway. Palmitate stimulated mitochondrial release of endonuclease G by a caspase 9-independent mechanism, thereby implicating endonuclease G in caspase 9-indpendent regulation of apoptosis by saturated FFA. We also observed that the unsaturated FFA oleate and linoleate prevented palmitate-induced mitochondrial release of both cytochrome-c and endonuclease G, which resulted in complete protection from palmitate-induced apoptosis.Taken together, these results demonstrate that palmitate stimulates apoptosis by evoking an intrinsic pathway of proapoptotic signaling and identify mitochondrial release of endonuclease G as a key step in proapoptotic signaling by saturated FFA and in the anti-apoptotic actions of unsaturated FFA. Recent evidence suggests that intracellular accumulation of saturated free fatty acids (FFA) in vascular cells contributes to lipid-mediated cellular damage , human active fragment caspase-8 and caspase-2 (BD Biosciences), endonuclease G , and β-Actin . Cell-permeable inhibitors of caspase 9 (Z-LEHD-FMK) and caspase-8 (Z-IETD-FMK) were from R&D systems . Etomoxir and 5-aminoimidazole-4-carboxamide-1-β-D-ribonucleoside (AICAR) were from Sigma and Toronto Research Chemicals , respectively.Fatty acid-albumin solutions were prepared by the protocol of Spector . BrieflyHuman mesangial cells (HMC), purchased from Cambrex Bioscience Inc. , were maintained in Dulbecco's modified essential medium (Gibco-BRL) supplemented with 17% fetal bovine serum (FBS), 100 U/ml penicillin, 100 μg/ml streptomycin, 5 ng/ml selenite, and 5 μg/ml each of insulin and transferrin. Characterization was performed by phase contrast microscopy and by immunostaining for intermediate filaments and surface antigens as described previously . Briefly3VO4, and 1 μg/ml luepeptin. After adjusting for cell protein , the amount of cleaved human caspase-3 (Asp 175) was measured by ELISA . For Western blotting of cleaved caspase proteins, the cells were washed with ice cold PBS and scraped in CHAPS extraction buffer and centrifuged at 2,000 × g for 10 min at 4°C. Protein content in the supernatant was assayed with the DC protein assay. An aliquot of the lysate (25 μg protein) was boiled in SDS sample buffer, resolved on a 4–12% SDS-PAGE gradient gel, and transferred to a 0.2 μm nitrocellulose membrane. After blocking in 5% non-fat dried milk in TBS-T  for 1 h, the membrane was washed 3 times with TBS-T for 5 min each and incubated overnight at 4°C with primary antibody in 3% BSA in TBS-T. After incubating with suitable HRP-labeled secondary Ab  and extensive washing, the proteins were detected by chemiluminescence with an average exposure ranging from 10–30 sec. As previously described [To measure endogenous levels of cleaved caspase-3, cells were lysed in a buffer containing 20 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 % Triton X-100, 2.5 mM Na pyrophosphate, 1 mM β-glycerophosphate, 1 mM Naescribed , the WesTo quantify the number of pyknotic nuclei, HMC on coverslips were washed once with PBS and fixed for 20 min with freshly-prepared 3.7% formaldehyde/20% sucrose in PBS. After washing twice with PBS the HMC were stained with 5 μg/ml Hoechst 33342  and mounted in Slow Fade Light (Molecular Probes). Using a Nikon Diaphot microscope, the number of pyknotic nuclei were counted and expressed as a percentage of the total number of nuclei counted (n = >300 nuclei per condition).in situ for 10 min at 200 × g and the supernatant gently removed. The monolayer was incubated in lysis buffer for 30 min at room temperature and centrifuged again at 200 × g. The supernatant  was assayed immediately for nucleosomal fragments.DNA fragmentation was assessed by measuring release of nucleosomal fragments into the cytosol . Briefly, HMC in 24-well plates were centrifuged HMC treated with FFA were lysed  and frozen at -40°C. Equivalent amounts of total HMC protein were added to buffer containing the LEHD caspase 9 peptide substrate linked to a cleavable luciferase substrate, aminoluciferin (Promega). The amount of light produced in a coupled reaction with luciferase was measured once every hour for 3 hours in a Berthold Luminometer. Experiments with increasing amounts of cell protein confirmed that the assay was in the linear range under the conditions described.To analyze cytochrome-c redistribution in HMC treated with FFA, cells were fractionated into cytosol and membrane fractions using 0.05% digitonin in an isotonic sucrose buffer exactly as described by Dong and coworkers . BecauseIntracellular accumulation of long-chain saturated FFA  has been shown to induce apoptosis in several cell types including cardiac myocytes and pancreatic β-cells ,11,13. TWe next investigated the signaling pathways by which palmitate induces apoptosis in HMC. To begin answering this question, we examined activation of initiator caspases associated with different pathways of apoptotic signaling. Cleavage of procaspase zymogens is required to form active heterotetrameric caspase complexes, so we analyzed the time course of caspase cleavage by Western blotting in 3 independent experiments. The p35 cleaved fragment of caspase 9 was elevated in cells treated with palmitate Fig. . After 4To confirm that palmitate activated caspase 9, we used a luminometric caspase 9 substrate to directly measure caspase 9 enzyme activity in cytosolic extracts of HMC. Palmitate at 0.4 mM increased caspase 9 activity 2.2- and 4.8-fold at 24 and 48 h, respectively  declare that they have no competing interests.RM carried out most of the technical studies and helped draft the manuscript. MSS conceived of the study, participated in its design and execution, and helped to draft the manuscript.


In neuropathological processes associated with neutrophilic infiltrates, such as experimental allergic encephalitis and traumatic injury of the brain, the CXC chemokine, macrophage inflammatory protein-2 (MIP-2) is thought to play a pivotal role in the induction and perpetuation of inflammation in the central nervous system (CNS). The origin of MIP-2 in inflammatory disorders of the brain has not been fully defined but astrocytes appear to be a dominant source of this chemokine.Curcumin is a spice principle in, and constitutes approximately 4 percent of, turmeric. Curcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation. Relatively unexplored, but relevant to its potential therapeutic efficacy in neuroinflammatory syndromes is the effect of curcumin on chemokine production. To examine the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its known anti-oxidant activities, we studied the effect of this spice principle on the synthesis of MIP-2 by astrocytes.Primary astrocytes were prepared from neonatal brains of CBA/CaJ mice. The cells were stimulated with lipopolysaccharide in the presence or absence of various amount of curcumin or epigallocatechin gallate. MIP-2 mRNA was analyzed using semi-quantitative PCR and MIP-2 protein production in the culture supernatants was quantified by ELISA. Astrocytes were transfected with a MIP-2 promoter construct, pGL3-MIP-2, and stimulated with lipopolysaccharide in the presence or absence of curcumin.The induction of MIP-2 gene expression and the production of MIP-2 protein were inhibited by curcumin. Curcumin also inhibited lipopolysaccharide-induced transcription of the MIP-2 promoter reporter gene construct in primary astrocytes. However MIP-2 gene induction by lipopolysaccharide was not inhibited by another anti-oxidant, epigallocatechin gallate.Our results indicate that curcumin potently inhibits MIP-2 production at the level of gene transcription and offer further support for its potential use in the treatment of inflammatory conditions of the CNS. As is true of other naturally occurring polyphenolic compounds, such as caffeic acid phenyl ester, rosmaric acid and resveratrol, curcumin possesses antioxidant properties which may reduce the production of free radicals and improve cell viability under conditions of enhanced oxidative stress. CurcumiCurcumin's immunomodulating and antioxidant activities suggest that it might be a useful adjunct in the treatment of neurodegenerative illnesses characterized by inflammation such as Alzheimer's disease. RelativTo explore the possibility that curcumin may influence CNS inflammation by mechanisms distinct from its antioxidant and known anti-inflammatory activities, we examined the effect of this spice principle on the synthesis of MIP-2 by astrocytes. Our results indicate that curcumin potently inhibits MIP-2 production at the level of gene transcription and offer further support for its potential use in the treatment of inflammatory conditions of the CNS.Six to eight-week-old CBA/CaJ mice were purchased from Jackson Laboratories  and bred in our animal facility.E. coli lipopolysaccharide  were purchased from Sigma, . Rabbit anti-cow glial fibrillary acidic protein polyclonal antibody was obtained from Dako Corp. .Curcumin, epigallocatechin (EGCG) and g. The pellet was suspended in Hank's Buffered Salt Solution (HBSS) and debris was removed by gravity sedimentation on ice for 3 min. The supernatant was collected, centrifuged and the pellet was washed twice with culture medium consisting of DMEM containing 10% heat-inactivated fetal bovine serum , 1 mM L-glutamate and penicillin/streptomycin . The cells were plated on 35 mm dishes and cultured at 37°C in a humidified atmosphere contain 5% C02. After 16 hours, plates were washed to remove non-adherent cells and debris. For experiments in which mRNA or MIP-2 protein were quantified, adherent cells were cultured until they reached confluence. For transfection experiments, adherent cells were cultured until they were nearly confluent. Medium was refreshed in all astrocyte cultures every 2–3 days. The preparations were >98% glial fibrillary acidic protein positive, as measured by flow cytometric analyses using a EPICS XL flow cytometer[Preparation and culture of astrocytes: Astrocytes were prepared from the brains of neonatal (3 to 7-day-old) CBA/CaJ mice by a modification of the method of Pousset et al. Brieflycytometer.4 M to 10-6 M) for up to 48 hours, the supernatants were then harvested and LDH was measured by colorimetric assay using a kit from Sigma diagnostics.Cell viability determination: The effect of curcumin on the viability of astrocytes was assessed by measuring cytosolic lactate dehydrogenase (LDH) leakage into the media as detailed earlier. Briefly4 M to10-7 M). After 4 hours of culture, cells were harvested and mRNA was isolated as previously reported[3M to 10-4M). To assess the effect of curcumin on MIP-2 protein production, astrocytes were cultured with LPS in the presence or absence of curcumin (10-5M) for 16 hours. Supernatants were then harvested and MIP-2 levels were determined by enzyme linked immunosorbant assay .mRNA and protein analyses: Confluent cultures of astrocytes were incubated with LPS (10 ηg to 5 μg/ml) for varying periods of time in the presence or absence of curcumin  DNA using the primers: 5'GCCCACCGAGTCTCTGTTTC3' (forward) and 5'GTTGGTGGCCAGCAGGAGGA3' (backward), then digesting with Rsa I/Nco I. The fragment, which corresponded to base pairs -539 to -2, relative to adenine (assigned +1) in the translation initiation codon of the MIP-2 gene (accession number AJ49888), was ligated to a Sma I/Nco I digested, promoterless luciferase reporter vector, pGL3-Basic . The direction of the insert was confirmed by restriction endonuclease digestion and its fidelity determined by sequence analyses as previously described. The MIP4M to 10-7M) was then added and transfected cells were further cultured for 24 hours. At the conclusion of culture, cells were harvested, cell lysates were prepared, and lysates were analyzed using a luciferase assay system  in accordance with the manufacturer's instructions.Transfection experiments: Astrocytes were transfected cells using a modification of the method of Franzoso et al. Briefly5M) that had no effect on viability as measured by LDH release (data not shown), abrogated LPS-stimulated MIP-2 production . Culturing such astrocytes with a dose of curcumin (10-5M) markedly inhibited chemokine gene expression in response to LPS . As was true for MIP-2 protein, culture of astrocytes with curcumin , were fused to a promoter-less luciferase reporter gene , induces the release of MIP-2 within 4 to 8 hours of o culture. Since T2 production in blood samples taken 1 hour after dose on days 1 and 29 of treatment by approximately 60%[Apart from its ability to inhibit MIP-2 production, curcumin's pleotropic antiinflammatory and anti-oxidative properties suggest its possible use in diseases of the brain accompanied by inflammation. Thus, LPS stimulation transcriptionally upregulates inducible nitric oxide synthase and cyclooxygenase-2 genes in microglia. This leads to the synthesis of nitric oxide (NO) and prostaglandins (PGs), respectively, and the possible formation of neuron-damaging free radicals, such as peroxynitrite. Curcumin abrogates the production of both NO and PGs in LPS activated microglial cells. In a reately 60%. Consistately 60%, and to ately 60%.In summary, the capacity of curcumin to inhibit astrocyte production of MIP-2, together with its broad immunosuppressive activities, strongly support the potential use of this spice principle in the treatment of inflammatory diseases of the CNS.EAE, experimental allergic encephalitis; EGCG, epigallocatechin gallate; LDH, lactate dehydrogenase; LPS, lipopolysaccharide; MIP-2, macrophage inflammatory protein-2; NFκB, nuclear factor kappa B; NO, nitric oxide; PG, prostaglandin;pGL3-MIP-2, a reporter gene construct containing the MIP-2 promoter; PMN, polymorphonuclear leukocyte; TBI, traumatic brain injury; TNFα, tumor necrosis factor alpha.The author(s) declare that they have no competing interests.MT participated in experimental design, acquisition of data, supervised all experiments, and carried out isolation of astrocytes, ELISA and transfection assays.BH isolated and amplified the MIP-2 gene promoter, and generated the MIP-2 promoter construct, pGL3-MIP-2.CS participated in culture of astrocytes and PCR analysis of MIP-2 gene.TS conceived of the study, participated in its design, and helped to draft the manuscript.All authors read and approved the final manuscript.


The commonest cause of end-stage renal failure (ESRF) in children and young adults is congenital malformation of the kidney and urinary tract. In this retrospective review, we examine whether progression to ESRF can be predicted and whether treatment with angiotensin converting enzyme inhibitors (ACEI) can delay or prevent this.2 with at least 5 years of follow up (median 143 months). 48 patients received ACEI. We explored potential prognostic factors that affect the time to ESRF using Cox-regression analyses.We reviewed 78 patients with asymmetric irregular kidneys as a consequence of either primary vesico-ureteric reflux or renal dysplasia , or abnormal bladder function . Patients (median age 24 years) had an estimated GFR (eGFR) < 60 ml/min/1.73 m2, mean proteinuria 144 (14) mg/mmol creatinine (1.7 g/24 hrs). Of 78 patients, 36 (46%) developed ESRF, but none of 19 with proteinuria less than 50 mg/mmol and only two of 18 patients with eGFR above 50 ml/min did so. Renal outcome between Groups 1 and 2 appeared similar with no evidence for a difference. A benefit in favour of treatment with ACEI was observed above an eGFR of 40 ml/min (p = 0.024).At start, mean (SE) creatinine was 189 (8) μmol/l, mean eGFR 41 (1) ml/min 1.73 mThe similar outcome of the two groups supports the nephrological nature of progressive renal failure in young men born with abnormal bladders. There is a watershed GFR of 40–50 ml/min at which ACEI treatment can be successful at improving renal outcome. Nearly half the children and young adults who develop end-stage renal failure (ESRF) have asymmetric irregularly shaped kidneys . This apSuch patients fall into two broad groups. Firstly, there is a group who appear to have normal bladders without outflow obstruction and normal calibre ureters when not micturating, described as having either primary VUR or primary renal dysplasia. Secondly, there is a group with some form of bladder outflow dysfunction which causes a secondary VUR and dilated upper urinary tracts, of which a posterior urethral valve (PUV) in males is the most common cause.99 mTc-dimercaptosuccinic acid (DMSA) renography .Hypertension was defined as either blood pressure consistently > 140/85 mmHg, or patients receiving blood pressure lowering therapy.End-stage renal failure (ESRF): was taken to be the date when the patient began dialysis. To calculate changes in renal function with time the eGFR was assumed to be 8 ml/min at this time.Outcome: Renal outcome was defined as having reached ESRF or not at last review.Since June 1986 some patients were started on ACEI therapy when anti-hypertensive therapy was required. In addition, some anti-hypertensive regimens were changed to ACEI therapy. A small group with blood pressure <140/85 were started on ACEI therapy because of increasing proteinuria. Some patients never received ACEI because ESRF was reached before the use of ACEIs in renal insufficiency had become routine.Patients, who were started on ACEI for hypertension, were advised to restrict their salt intake and the initial aim was for a blood pressure of ≤ 130/70. If ACEI alone did not lower the blood pressure to the target a diuretic was added. The latter was not always possible in Group 2 patients who might already have features of hypovolaemia secondary to their renal tubular pathology. No patient ever received any immunosuppressive drug.Only data from the start and end of the study are presented from all 78 patients. Of the 48 patients who were treated with ACEI, we also report data on 28 of the patients (for whom it was available for at least 18 months before and 48 months after the introduction of ACEI.) at the start of ACEI therapy and again at 2 years after start of therapy.Means were compared within groups by paired samples t-test, and between groups by Wilcoxon rank-sum test. The time scale was time since birth, since the setting was of a congenital disease eventually leading to renal failure. It was checked graphically that there was no obvious pattern to loss to follow up over time. Renal outcome (reaching ESRF or not) was compared between groups using Kaplan-Meier survival plots. Outcomes were quantified as the median survival outcome in months . This is equivalent to the median time for 50% of the group to reach ESRF. Differences in renal outcome over time were tested using log-rank tests. For graphical examination of the proportionality, continuous variables were grouped into approximate tertiles. The Cox proportional hazards model was chosen for further analysis. Continuous variables were centred on the mean. Protein/urine creatinine ratios were log-transformed for further analyses. Treatment with ACEI was entered as a time-changing variable to take account of the variable start of treatment after referral. Univariable regression models estimated crude (unadjusted) effects of the prognostic variables. Since it is possible that the effect ACEI depends on the GFR at start of treatment (entered as continuous variable), especially in patients with moderate to severe renal insufficiency, an interaction between those variables was fitted. Variables were entered in the multivariable model in a stepwise forward fashion. Analyses were performed using SPSS for Windows v10.1. and Stata 8 .Data from 78 patients, who were first seen between December 1969 and February 1988, were analysed. Demographic details are presented in Table Group 1  were patients who had primary VUR or primary renal dysplasia. Twenty two patients  presented in childhood with a UTI. In each case, MCU performed at a median age of 7 years showed reflux which was either bilateral (82%) or unilateral (18%). In contrast, the remainder (n = 22) presented at a median age of 24 years. Only 32% were female and they almost invariably presented either with hypertension after starting a contraceptive pill or with complications during pregnancy. Only 2 had had a MCU performed and neither showed VUR.Group 2 patients  had the following diagnoses: PUV (n = 15), prune belly syndrome (n = 2), single dysplastic kidney with megaureter (n = 2), renal dysplasia with abnormal bladder function (n = 2), bilateral megaureters (n = 1), megacystis and megaureters (n = 4), and finally a group (n = 8) in whom the initial diagnosis (pre-1979) had included "bladder neck obstruction". Six of these had megacystis and megaureters and might now be termed `pseudo-prune belly syndrome'.By Group: The median survival time of Group 1 versus Group 2 was compared by log rank test: 231 months (95% CI: 153–309) vs. 162 months (95% CI: 135–189) respectively, p = 0.35. There was no evidence for a major difference in renal outcome between these patients with primary and secondary reflux. Thus for subsequent analyses the data from all 78 patients was combined.By eGFR: We compared renal outcome for all patients after they were stratified by initial eGFR into four groups    after the start of the study, by which time their median eGFR had fallen from 46 (range 15–60) to 36 (10–60) ml/min/1.73 mTable We examined the effect of ACEI on 28 patients with deteriorating function for whom data was available for at least 18 months before and 48 months after the introduction of ACEI. ACEI reduced the rate of loss of renal function during the first 24 month period of follow-up, but the benefit was greater in the subsequent follow-up period with the rate slowing from a median of -1.86 before ACEI to -1.48 ml/min/yr (p = 0.007). ACEI treatment was associated with a reduction in proteinuria after 24 months of therapy. However, proteinuria had increased at last follow up owing to loss of the anti-proteinuric effect in half the group Fig .Analysis of 30 patients who did not receive an ACEI provides indirect support of benefit from this treatment. The median rate of loss of renal function for 25 of these patients who started with a eGFR ≤ 50 ml/min was 2.80 ml/min/yr and if initial proteinuria was ≥ 50 mg/mmol, (n = 23) the rate of loss of renal function was 3.0 ml/min/yr.At the start of the study 18 (41%) Group 1 patients and 5 (15%) Group 2 patients were hypertensive. Group 2 patients tended to be normotensive and some were started on an ACEI for proteinuria. At ESRF or last follow up, all patients were on conventional anti-hypertensive therapy or ACEI, except for two Group 2 patients.and proteinuria have progressive renal failure, that the rate of decline of function is proportional to the magnitude of the proteinuria, and that angiotensin antagonists both slow the rate of progression and reduce proteinuria [There is a consensus that patients with renal insufficiency teinuria -20. Our While VUR patients with abnormal bladders almost invariably present in early childhood, patients with normal bladder function have a bimodal presentation. In one series from New Zealand, 42 patients (36 adults) had ESRF with reflux nephropathy. Many had presented with advanced renal insufficiency, hypertension, and proteinuria, and only 22% of males and 58% of females had a history of UTI. Similarly, in our study, VUR had been proven in 50% of patients with primary reflux nephropathy and these patients  had almost invariably presented with an UTI in childhood (median age 7.0 years). In contrast, the other half  presented at a median age of 24 years with advanced disease and persistent reflux was not demonstrated in the 2 patients who underwent a MCU. Similar to the New Zealand experience ,22, nearAlthough reflux nephropathy is frequently viewed as a disease of little girls with recurrent UTIs , our datIn 1978 Kincaid-Smith and her colleagues  reportedIn an Italian study , 80 patiIn a UK study from Newcastle, proteinuria and renal insufficiency (plasma creatinine >130 μmol/l) were present from presentation in 21% and 13% of 125 patients respectively, and with time (mean 5.9 years) a further 21% developed proteinuria and 22% renal insufficiency. In all the 16 patients with progressive renal failure, the decline was linear. In a subsequent report, progressive renal failure did not develop in 138 adult patients with normal function at the start (plasma creatinine < 90 μmol/l) [2 nephropathy rarely progresses, but disease progression is invariable when function is worse. Nakashima et al. similarly reported that an isotopic GFR less than 49 ml/min predicted decline to ESRF [Our data support the established relationship between the risk of progressive nephropathy and renal insufficiency with proteinuria, but suggests a watershed range for renal function as a predictor of outcome. When the eGFR exceeds 40–50 ml/min/1.73 m to ESRF .The other determining factor of poor renal outcome is proteinuria, and we find that deterioration can be expected when proteinuria exceeds 50 mg/mmol (0.5 g/d). In a Japanese study, serial biopsy samples from patients with reflux nephropathy confirmed the close association between the degree of renal scarring, the extent of the glomerular pathology, and proteinuria ,26. Once2 at outset [In a 10-year follow up study of 52 children, randomised to medical or surgical management of severe bilateral VUR (grades III-IV) between 1985–1989, progressive renal failure developed in only 4 children (2 from each group) all of whom had GFRs at or below 40 ml/min/1.73 mt outset .Despite the few long-term studies of adult patients with primary VUR and reflux nephropathy -5, therein utero may not prevent progressive renal damage [absence of proteinuria, then some other cause (such as obstruction) must be sought.Progressive renal damage due to congenital outflow tract obstruction may be averted by urological intervention. This is not, however, always successful and even treatment l damage  and the l damage . Most bol damage  althoughApart from small numbers, the limitations of this study are the usual ones in observational research with effect estimates that are potentially confounded. We believe, however, that it would not now be possible to design a prospective study in which ACEI therapy was with held from one group. In this study, ACEI therapy improved renal outcome although non-ACEI patients were generally from an earlier period and not necessarily seen in a specialist clinic. The analysis demonstrated a long-term benefit for ACEI both in slowing the rate of loss of function and in reducing proteinuria. However, ACEI therapy appeared to be ineffective if started when the eGFR was already = 30 ml/min.We have studied a group of patients whose renal pathology is not immunological and whose residual functioning tissue is not homogenously distributed. The pathophysiology is similar to experimental sub-total nephrectomy which results in proteinuria, progressive glomerulosclerosis and renal failure ,31. LikePatients with a GFR of <60 ml/min need careful follow up and we would recommend that anyone with increasing proteinuria, or proteinuria >50 mg/mmol (0.5 g/d) is started on an angiotensin antagonist to reduce proteinuria and slow the rate of progression of the renal failure.GHN has been a consultant and lectured for MSD regarding both ACE inhibitors and angiotensin II receptor blockers. G Thomson, D Nitsch, RG Woolfson, JO Connolly, and CRJ Woodhouse have no conflict of interest.GN was responsible for the study design, management of patients, and writing of the report. GT collected clinical data from medical records; DN contributed to analyses and writing of the report; RW and JC contributed to patient management and writing of the report. CW was responsible for the continuing urological supervision of the patients and writing of the report. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Fibrin D-dimer levels have been advocated as an useful clinical marker of thrombogenesis.We hypothesized that i) there is a hyperclotting state after the return of atrial fibrillation to sinus rhythm, ii) the measurement of plasma D-Dimer levels might be a good screening tool of this clotting status, and iii) the duration of arrhythmia influences the haemostasis measured by plasma D-Dimer levels.Forty-two patients with atrial fibrillation undergoing cardioversion were divided into two groups: in Group A  the duration of atrial fibrillation was 72 hours or more (142.7 ± 103.8 hours), in Group B  the duration of atrial fibrillation was less than 72 hours (25 ± 16 hours). Plasma fibrin D-dimer levels were measured by enzyme immunoassay before, and 36 hours after, cardioversion. The change of plasma D-dimer levels 36 hours after cardioversion was calculated as delta-D-dimer.There were no significant differences in demographic, clinical, and echocardiographic data, and the success of cardioversion between the two groups. Compared to the control, the baseline D-dimer levels were significantly higher in both groups. The delta D-dimer levels were significantly higher in Group A than in Group B (p < 0.005). Furthermore, plasma D-dimer levels 36 hours after cardioversion  and delta-D-dimer levels  showed significant correlations with the duration of atrial fibrillation.The longer duration of the atrial fibrillation episode could lead to a more prominent cardiovascular hyperclotting state after cardioversion, and the mean changes of plasma D-Dimer levels could be used as an useful clinical marker of the clotting state after atrial systole return. Atrial fibrillation is the most common sustained arrhythmia in clinical practice. It is associated with an increased risk of thrombus formation, resulting in substantial morbidity, with the augmented risk of stroke being the most serious. This could be explained by haemostasis conditions favouring thrombosis: previous studies have demonstrated that in most patients, AF is a high risk factor for hypercoagulability, irrespective of underlying structural heart diseases or aetiology -8.In addition, it is well known that the direct current cardioversion of atrial fibrillation, especially if persisting >48 h, carries a great risk of thromboembolism, which extends to 5% of cases not receiving anticoagulant therapy. The mechanism and pathogenesis of thromboembolic episodes after restoration of sinus rhythm in these patients is not completely understood.There is some evidence that the prothrombotic state associated with atrial fibrillation might contribute towards the risk of thromboembolism following cardioversion, but reports are not clear. In this context, having a marker of coagulation activation would be useful in identifying patients at highest thromboembolic risk. Indicators of hypercoagulability, such as D-dimers, which are indicative of a prothrombotic state, might also be indicative of thromboembolic risk . D-dimerWe therefore hypothesized that i) there is still a hyperclotting state after return of sinus rhythm, ii) the measurement of plasma D-dimer levels pre- and post-cardioversion might be a good screening tool of this clotting status, and iii) the duration of arrhythmia could be a good predictor of thromboembolic events after cardioversion, due to the influence on haemostasis measured by plasma D-dimer levels.Over a period of 18 months, we studied 42 consecutive patients, aged between 39–68 years old, with non-valvular atrial fibrillation who underwent successful electrical cardioversion and who remained in sinus rhythm at the one-month visit. Exclusion criteria were other acute causes of atrial fibrillation , acute cardiovascular or cerebro-vascular events  occurring within five months, valvular heart disease, malignancy, connective tissue disease, infectious or inflammatory conditions and chronic renal/hepatic disease. The patients were divided into two groups. In Group A , the duration of atrial fibrillation was 72 hours or more (142.7 ± 103.8 hours). In Group B  the arrhythmia had a duration of less than 72 hours (25 ± 16 hours). We included only patients treated with anticoagulant-coumadin for chronic prophylaxis. There was not a statistically significant difference between the mean duration of anticoagulation treatment between the two groups (14 ± 4 months for group A and 12 ± 6 months for Group B). Prothrombin time to an INR  of 2.5–3 was considered as a necessary inclusion criterion for all patients. The total population had a history of drug refractory atrial fibrillation, with a serious number of arrhythmia episodes. We only analyzed the documented arrhythmia episodes. The total number of documented episodes of paroxysmal atrial fibrillation in Group A was 33, and in Group B the patients had 36 confirmed arrhythmia episodes. There were no significant differences in age, sex, hematocrit, hemoglobin, plasma fibrinogen level, underlying heart disease, success ratio of electrical cardioversion, echocardiographic data, presence of diabetes mellitus or hypertension between the two groups. The clinical characteristics of the patients are shown in Table An intravenous line was placed and blood samples for D-dimer measurement were taken from the patients immediately before cardioversion, and 36 hours after recovery of sinus rhythm. Citrated plasma was obtained from venous blood by centrifugation at 2,500 rpm for 15 min at 4°C. Aliquots were stored at -70°C to allow batch analysis. The plasma D-dimer levels were measured by the enzyme-linked immunosorbent assay method. The measurements were obtained with the use of a quantitative sandwich immunochromatographic technique . For every blood sample, measurements were done twice. The investigators and attending physicians were blinded to the D-dimer test results. Inrta-assay coefficients of variation for assays were <5%, inter-assay variances were 10%. The changes of plasma D-dimer levels 36 hours after cardioversion were calculated as delta- D-dimer.Echocardiographic examinations were performed in all patients, immediately prior to the procedure and 36 hours after successful cardioversion. Transthoracic echocardiographic two-dimensional imaging and guided pulsed wave Doppler recordings were obtained. Transmitral Doppler inflow velocities were recorded from the apical four-chamber view.Peak velocities of early fillings (E) wave and atrial filling (A) wave were determined. The inter- and intra-observer variability was <5% for these measurements. We did not detect the presence of thrombi in the left atrium. However, the absence of thrombus on transthoracic echocardiography does not preclude the real absence of thrombus.Clinical variables are expressed as the mean value ± SD. The effects of duration from the onset of atrial fibrillation on the measured indexes were analyzed by two-way repeated measures analysis of variance (ANOVA). Sequential data pre- and post-cardioversion were analyzed by Friedman's repeated measures analysis of variance. Correlations were performed by Spearman's rank correlation method. Stepwise multiple regression analyses were performed to determine independent predictors for plasma D-dimer levels, using age, sex, left-atrial size, left ventricular dimensions  presence of underlying medical disease, smoking status, and the presence of atrial fibrillation. A p value < 0.05 was considered statistically significant. Analyses were performed with SAS for Windows 8.02  and GraphPad Prism, version 3.00  statistical software packages.The baseline D-dimer levels of each group are shown in Table The patients' sinus rhythm was restored by applying electrical cardioversion in all study patients. Delta D-dimer levels were significantly higher in Group A than in Group B. (p < 0.005).There were no significant differences in plasma D-dimer levels before, and 36 hours after, cardioversion between the two Groups .Two embolic stroke events were recorded during the six hours after cardioversion in Group A  and none in Group B. The embolic events occurred in two women with an INR of 2.6 and 2.9 respectively. At the onset of the event one patient had D-dimer levels of 112.2 ng/ml while the second patient had D-dimer levels of 109.8 ng/ml. Fortunately, these were transient events. The computerized tomography detected non-extentend thromboembolic areas.In the present study we analyzed D-dimer levels as a screening index of a hyperclotting state after cardioversion of atrial fibrillation. It would be more useful if we used the term abnormal haemostasis, because the fibrin D-dimer is a cross-linked degradation product, resulting from the balance between thrombogenesis and the fibrinolysis process.Fibrin D-dimer levels have been established as a useful clinical marker of thrombogenesis.  The use In "normal" patients, elevated D-dimer levels have been associated with a higher risk of developing cardiovascular disease.  Also, suPatients with long-lasting episodes of atrial fibrillation or chronic atrial fibrillation are characterized by increased levels of plasma D-dimers ,22, platIn the present study, the plasma D-dimer levels decreased in both groups after successful cardioversion, but the mean change in D-dimer levels pre- and post- cardioversion was significantly lower in Group A than in Group B, and D-dimer levels continued to be high, even 36 hours after the procedure in Group A. There were reported embolic events in 8.33% of Group A patients, with the higher delta D-dimer.These data confirm the existence of an abnormal clotting state after the cardioversion of atrial fibrillation, which could be the cause of the thromboembolic events observed after the return to sinus rhythm, even under anticoagulation.The mechanism of thromboembolism is debatable. The results we found could suggest an embolism might be caused by detachment of a formed thrombus during the phase of passage from AF to sinus rhythm, due to being heavily compromised by stunning the mechanical function of the left atrium and left atrium appendage after sinus rhythm restoration. The stunning of the left atrium and left atrium appendage might attenuate the thrombogenetic activation which our result showed.In the present study we also provided evidence that this hypercoaguable state after successful cardioversion of atrial fibrillation related directly to the duration of the arrhythmia episode. It has been accepted that chronic atrial fibrillation is characterized by increased levels of plasmatic D-dimers, with a wide inter-individual variability, depending on the patients and therapeutic characteristics. However, it has not been established if this level could also be characteristic of paroxysmal or persistent atrial fibrillation in patients, and whether it could be a predictive factor for the risk of thromboembolic events after cardioversion to sinus rhythm.Our results show that longer duration of atrial fibrillation could lead to a more prominent cardiovascular hyperclotting state after cardioversion and that the duration of the arrhythmia episode might be a risk factor for the high incidence of post-cardioversion thromboembolic events. Importantly, this hypercoaguable state does not appear to be subject to any other clinical variables, nor is it related to whether or not the patient had a lone atrial fibrillation or not, suggesting that the duration of the arrhythmia episode confers a constant prothrombotic state per se, after cardioversion to sinus rhythm, which was independent of the etiology but dependent on the duration of the arrhythmia. Interestingly enough, this hyperclotting state after cardioversion exists even under appropriate anticoagulative treatment. The relation of the markers of accelerated coagulation to clinical or echocardiographic risk factors for thromboembolism is controversial.Our results clearly demonstrate a positive correlation of the based on the arrhythmia duration clinically predicted embolic risk to the mean change of plasma D-dimer levels 36 hours after successful cardioversion. We could not detect any relation to the echocardiographic risk factors, probably due to our inability to perform a transesophageal echocardiography. The present study suggests that even in the absence of clinical conditions causing increased embolic risk, patients with signs of accelerated coagulation are at risk of thrombus formation in the future.In conclusion, a longer duration of the atrial fibrillation episode could lead to a more prominent cardiovascular hyperclotting state after cardioversion, and the mean changes of plasma D-Dimer levels could be used as an useful clinical marker of the clotting state after atrial systole return.Our study might have practical implications for the management of patients with an episode of atrial fibrillation, regardless of the anticoagulative treatment they are receiving: the episode must be terminated as soon as possible, because the pathogenesis of thrombus formation in atrial fibrillation is very complex and not yet completely defined. Further investigations with a large population are needed to define all the pathophysiologic mechanisms of thrombus formation.