All organisms have evolved complex mechanisms designed to exquisitely regulate the expression of appropriate genes at their correct levels. Natural random variation in the processes of regulation and expression, however, limits the precision with which protein production can be controlled. This subtle variation, or “noise,” in the expression of genes has been studied with increasing interest. Though much progress has been made in understanding the amount of noise that exists and the cellular processes that underlie it, the physiological impact of noise, and whether it is biologically relevant or can just be ignored, has been less clear.To answer this question, Hunter Fraser et al. asked whether noise in gene expression exerts an equal effect on all genes in the genome. Is noise in gene expression irrelevant to the fitness and well-being of cells, or do cells need to minimize noise in the expression of some or all genes? If noise in gene expression has a negative impact on cells, they reasoned, that impact should vary from gene to gene depending on the gene's function. There should be selection to minimize noise for those genes most crucial to cell survival and function. Thus, a genome-wide analysis of noise in gene expression, they predicted, would show that genes for which “noisy” expression would be most harmful would display less of it.Saccharomyces cerevisiae because of the vast quantity and variety of genomic data available for this organism. Previous research has shown that the noise that exists in the expression of a gene is directly related to the rates of transcription and translation. Using data available from previous genome-wide studies, the authors were able to estimate these rates, and therefore the noise, for nearly every gene in the yeast genome.The researchers examined this question in the budding yeast After estimating the amount of noise in the expression level for nearly every gene, the authors examined two subsets of genes that they hypothesized would be particularly affected by noise. First they looked at “essential” genes, reasoning that since total lack of expression of these genes results in death, even small variations in expression resulting from noise would often exert a negative impact. Previous research had identified all the essential genes in yeast by deleting each gene individually and assessing the fitness of the resulting mutant. Here, the authors compared the levels of noise in this pool of essential genes to that of nonessential genes. They found that essential genes usually display less noise than nonessential genes, lending support to their hypothesis.They similarly examined genes encoding proteins involved in forming multiprotein complexes. Because these complexes are built of proteins in specific ratios, over- or under expression of one component will hinder the accurate assembly of productive complexes. So a high degree of noise could interfere with the coordinated expression necessary for proteins involved in these complexes. The authors again used data from previous research to choose members of this group: they relied on two studies which had identified a large number of multiprotein complexes in yeast. Using this group of genes for comparison, the authors found that, like essential genes, genes encoding proteins involved in multiprotein complexes generally display less noise than other genes.This study draws a simple but fundamental conclusion about noise in eukaryotic gene expression—noise has physiological consequences. Importantly, the fact that noise is minimized in those gene groups for which noisy expression would be most harmful suggests that factors contributing to noise are subject to natural selection. This study also demonstrates the power of using the growing number of genomescale datasets in this type of analysis. Researchers will undoubtedly continue to mine the available data to draw biological conclusions not anticipated by the original authors.


Loa loa has recently emerged as a filarial worm of significant public health importance as a consequence of its impact on the African Programme for Onchocerciasis Control (APOC). Severe, sometimes fatal, encephalopathic reactions to ivermectin (the drug of choice for onchocerciasis control) have occurred in some individuals with high Loa loa microfilarial counts. Since high density of Loa loa microfilariae is known to be associated with high prevalence rates, a distribution map of the latter may determine areas where severe reactions might occur. The aim of the study was to identify variables which were significantly associated with the presence of a Loa microfilaraemia in the subjects examined, and to develop a spatial model predicting the prevalence of the Loa microfilaraemia.Loa loa microfilaraemia using individual level co-variates (age, sex, μl of blood taken for examination) and village level environmental co-variates .Epidemiological data were collected from 14,225 individuals living in 94 villages in Cameroon, and analysed in conjunction with environmental data. A series of logistic regression models  was developed to describe variation in the prevalence of Loa loa prevalence was created within a geographical information system. The model was then validated using an independent data set on Loa loa distribution. When considering both data sets as a whole, and a prevalence threshold of 20%, the sensitivity and the specificity of the model were 81.7 and 69.4%, respectively.A spatial model of Loa-related severe adverse events. It is now routinely used by APOC when projects of community-directed treatment with ivermectin are examined.The model developed has proven very useful in defining the areas at risk of post-ivermectin  Loa loa is important in countries involved in the African Programme for Onchocerciasis Control (APOC) and is also now a significant issue for the Global Programme to Eliminate Lymphatic Filariasis (GPELF). Both programmes rely on the wide-scale distribution of anti-helminthic drugs to poor communities using community-directed drug distribution schemes. A problem was first observed in Cameroon where a series of reports of severe and sometimes fatal encephalopathic reactions to ivermectin (Mectizan®) in individuals with high Loa loa microfilarial counts was made [Loa loa endemic areas although evidence for this is contradictory [Knowledge of the spatial distribution of was made -3. Similadictory -7.Loa loa is associated with tropical "eye worm" (migration of adult worms across the sub-conjunctiva), Calabar swelling, oedemas and prurities but is not considered as pathogenic as other filarial worms, and is consequently less well studied. It is transmitted by species of horse-flies (Chrysops spp.), most commonly Chrysops dimidiata and C. silacea which inhabit the forest areas of West and Central Africa, extending to the Ethiopian border. Severe and fatal reactions to ivermectin have been associated with individuals with high Loa loa microfilarial loads (more than 30,000 microfilariae (mfs) per ml of blood) and those with more than 50,000 mfs/ml are considered at very high risk [igh risk ,8-10.Loa loa remains unclear [Changes in the protocols for drug administration and post-treatment surveillance in areas considered to be at-risk of severe reactions to ivermectin have been implemented ,12, but  unclear . Such inLoa loa [Loa loa infection may be high enough  that poses an operational problem for drug distribution by the Community-Directed Treatment with Ivermectin (CDTI) strategy [Recent studies have shown that there is a close relationship between intensity of microfilariae infection and prevalence rates of Loa loa ,15 suggestrategy .Loa loa in West and Central Africa based on the relationship of crude Loa loa prevalence data (obtained from a literature search) to a wide range of environmental variables. Initial results suggested that land/forest cover derived from NOAA-AVHRR satellite data , and soil type (from the FAO digitised soil map of Africa), are significant predictors and a preliminary risk map was produced [As a preliminary step we created a risk model for produced . Howeverproduced . PossiblInstitut de Recherche pour le Développement (IRD) at the Centre Pasteur du Cameroun (CPC). A second independent data set, collected as part of a project supported by the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases (TDR) to calibrate a rapid clinical diagnosis of Loa loa (RAPLOA) [In order to improve the quality of epidemiological data being used to develop the model, we created a new prevalence database from a series of surveys conducted by the team of the (RAPLOA) , was useLoa loa parasites in the blood of individuals was assessed. The data come from five regions: (a) the forest-savannah mosaic area of the Mbam and Kim division ; (b) the degraded forest area of the Lekie division ; (c) the dense forest area of the southern part of the Central province; (d) the highland savannah area of the Western Province; and (e) the savannah areas of the districts of Banyo and Bankim, in the Adamaoua province. The Ministry of Public Health of Cameroon provided ethical approval for these surveys.Epidemiological data used to create the model was obtained from a series of field studies undertaken by the IRD-CPC laboratory in Cameroon during the period 1991–2001 in which the presence of Loa mfs was recorded.The total number of subjects included in the analysis was 14,225 individuals for 94 villages. Study participants consisted of individuals over the age of 5 years who gave their consent or for whom consent was obtained from the parent or guardian. Individuals who had had filaricidal drugs, such as ivermectin or diethylcarbamazine, in the previous five years were not included in the study. The arrival of the research team in the villages was announced to the population one week before the event through the local authorities and teachers. The objective of the examinations were clearly explained, and it was stated that each individuals results would be returned. On the given day, the team settled at a central place of the village , and all the volunteers were examined between 10.00 and 16.00 hours. A standardized quantity of capillary blood was obtained with a non-heparinized micro-capillary tube. Prior to 1994, the amount taken was 30 μl, and thereafter, 50 μl. After Giemsa staining, the slides were examined under a microscope and the presence of Loa loa infection). A summary of the epidemiological data used is presented in Table The latitude and longitude of all study villages were obtained from either the ordinance survey map or a global positioning system. Thus the data set included village name, longitude and latitude, alongside data on individuals examined , we investigated whether some variables describing the environment of their place of residence (see list of variables below) were significantly related to the microfilaraemia. For these environmental variables, distance operatives indicating whether or not villages were within 5 km of potential at-risk variables were created. Distance operatives were based on the normal dispersal range of Chrysops which has been shown to be within 5 km of their breeding sites [The aim of the study was to identify variables which were significantly associated with the presence of a ng sites .Loa loa mfs in the individual. Then we developed a series of logistic regression models  to describe variation in the prevalence of Loa loa microfilaraemia using individual level co-variates (age, sex, μl of blood taken for examination) and village level environmental co-variates. Logistic regression is useful to predict the presence or absence of a characteristic or outcome based on values of a set of predictor variables. It is similar to a linear regression model but is suited to models where the dependent variable is dichotomous. Logistic regression coefficients can be used to estimate odds ratios for each of the independent variables in the model. In our example the dichotomous variable is the presence or absence of Loa loa infection in each individual in the sample. Those variables which did not have a significant relationship with age and sex adjusted prevalence were excluded from further analysis. All variables were analysed at individual level.In order to design a valid modelling structure, univariate analysis was first undertaken of the relationship between, on the one side, individual and environmental variables, and on the other side the variable of interest, i.e. the presence/absence of The distribution of the human population in sub-Saharan Africa is relatively well described from population census data obtained at the national and sub-national level. The population data for 1990 used in this analysis was obtained from the Global Resource Information Database of the United Nations Environment Programme (UNEP-GRID), Eros Data Centre . These pThe USGS  hydrologic digital dataset provides detailed descriptions of the topography of the area, including elevation , slope, aspect (direction of maximum rate of change in elevation between each cell and its eight neighbours and representing direction of slope), flow accumulation (defining amount of upstream area draining into each cell) .Satellite data indicating the 'greenness' of the environment  was obtained from the VEGETATION sensor launched in 1998 onboard the SPOT 4 satellite system. NDVI images have been specifically tailored to monitor global land surfaces' parameters on a daily basis with a medium spatial resolution of one km . They peIn order to take into account the flight distance of the vector, 5 km buffers were created around each study village and the mean value of the NDVI variable was obtained and used in the development of the logistic regression model.Loa loa endemicity [Loa loa mfs. Model validation was undertaken through correlating model outputs with the independent data set.Epidemiological data used to validate the model was obtained from a multicentric study supported by TDR which was designed to assess the relationship between clinical and parasitological indicators of demicity ,26. The Loa loa prevalence when regressed against population density was not found to be significantly correlated, a strong and significant linear relationship  was found with age below 40 years  higher in males than in females (19.8% n = 6725 and 15.2% n = 7500 respectively). Sex was therefore entered  in the logistic regression model.Prevalence rates were significantly (P < 0.001) in individuals from whom the blood smear was prepared using a volume of 50 μl as compared with those from whom 30 μl of blood were taken. Blood sample size was therefore entered  in the logistic regression model.As one would expect, prevalence rates were significantly higher  in predicting microfilaraemia prevalence using univariate and multivariate regression statistics, the DEM alone was chosen as a predictive variable for the development of a logistic regression model. Because the relationship between r Figure  the DEM Since univariate analysis of the SPOT VEGETATION NDVI data against prevalence indicated a non-linear relationship Figure  and 3b, Separate and combined models were created which included all individual level co-variates and altitude for both buffered and non-buffered SPOT VEGETATION data variables. Final model choice was based on (a) the simplicity and biological validity of the model (b) the predictive capacity of the model when assessed using bootstrap methodology  and (c) the ability of the model to be extended over the large geographic area involved in the APOC programme.Loa loa is represented by:Thus, besides the three individual-level covariates (sex, age and blood sample size), only three village level environmental co-variates were kept in our model: maximum annual NDVI, standard deviation of the NDVI, and elevation. In the final model, the probability of a female individual being infected with z-), where :1/(1+e[Z] = ([Sex]*0.221)+ ([Age] * 0.049)- ([Blood sample size]*0.542)2] * 74.38)+ ([Maximum NDVI3] * 58.816)- ([Maximum NDVI+ ([Standard deviation NDVI] * 11.788)+ (([Elevation] < 250) * 1.133)+ (([Elevation] = 250-500) * 0.865)+ (([Elevation] = 500-750) * 0.981)+ (([Elevation] = 750-1000) * 1.171)- (([Elevation] = 1000-1250) * 0.623)- (([Elevation] = 1250-1500) * 1.516)- (([Elevation] = 1500-1750) * 1.342)- (([Elevation] = 1750-2000) * 0.669)- 22.883 (constant),where [Sex] = 0 for females, and 1 for males; and [Blood sample size] = 0 for 30 μl, and 1 for 50 μl.2; it decreases (-) with Maximum NDVI3 (indicating that the relationship to maximum NDVI is non linear), and increases for categories of elevation between 0 and 1000 metres after which it decreases. The probability of infection decreases with increasing value of blood sample size (0 versus 1) even if larger blood sample should normally allow better detection of the infection. This could be due to the fact that larger samples have been taken often in places where infection rates are low, in the highlands, and this is presumably a result of interacting with the other variables.Thus for our example, age, sex and blood sample size variables are presented for a fixed value because of their non-spatial nature. The probability of infection increases (+) with increasing value for sex (0 versus 1), with increasing age, and with increasing standard deviation of NDVI and Maximum NDVIr2 of 0.6033 was obtained  were found to be 77.1 and 80.0%, respectively  and then tested on the remaining 50% of the data. When the model results were plotted against the observed data an d Figure . In addiIn order to standardize the variables relating to individuals (which cannot be mapped) we chose values that best represent the average adult population (above the age of 15) i.e. 50:50 male – female ratio, age 40 and a blood sample of 50 μl. Age 32 was the approximate average age of individuals in the data sets used to develop the model. Using age 40  in the model was therefore likely to slightly over-represent infection in these data sets but the final model would be directly comparable with current policy to exclude individuals below the age of 15 from rapid epidemiological surveys for loiasis ,15. In oChrysops infested area nearby. The three other villages  are all villages from the Eastern Province of Cameroon which were sited on very localised areas where model predictions for <20% prevalence occurred. These villages were all within 1 km of areas where model results predicted >20% prevalence.Since the chosen model was based on environmental variables, which could be mapped across the entire country, it was possible to extrapolate the model results to the whole of Cameroon Figure  and compWhen one considers both datasets as a whole, and the prevalence threshold of 20%, the sensitivity and the specificity of the model were 81.7 and 69.4%, respectively.®) has been targeted at areas of hyper- and meso-endemicity, identified by Rapid Epidemiological Mapping [Loa loa. This has led to caution in defining new areas for the expansion of the programme and a requirement for identifying areas of high risk of Loa loa using a potentially rapid and extensive methodology. Thomson et al. [Detailed information on distribution of disease and levels of endemicity is a prerequisite for effective planning of control programmes. This has been an important element in the planning of the African Programme for Onchocerciasis Control (APOC) where community-directed treatment with ivermectin  while the 1 km resolution of the environmental data cannot reveal the small and localised muddy breeding sites of the Chrysops vectors, their general habitat is well captured by the model and it is recognised that the 1 km resolution is much less than the dispersal capacities of the adult flies; and (b) the model is not dependant on the seasonal or inter-annual variability of the vector density, since this is not reflected in fluctuations in Loa microfilaraemias, because the adult worm life-span is much longer (4–17 years).This paper further develops the satellite-derived risk map of Loa loa are particularly high, according to the Environmental Risk Map (ERM), two should be pointed out, because they are also those where most of the cases of serious adverse events (SAEs) have been recorded so far. The first one is the Lekie Department , in the Sanaga valley, where 53 of the 63 probable or possible Loa loa encephalopathy cases recorded between 1989 and 2001 in Cameroon have occurred [Loa "strain" [Among the areas in which the prevalences of "strain" ,29. The While the use of satellite-derived environmental data for evaluation of disease risk has been developed for several vector borne diseases  , limitedLoa loa endemic areas". Three types of mass treatment strategies have been defined, according to the levels of endemicity of onchocerciasis and of loiasis, the latter being defined by the prevalence of Loa microfilariae (<20 % versus ≥ 20%) or of history of eye worm passage (<40 % versus ≥ 40%). In those areas where onchocerciasis is meso- or hyperendemic, and the prevalence of Loa mfs exceeds 20%, a number of detailed measures should be taken, regarding training of community distributors and medical personnel, availability of medical supplies, duration of distribution, surveillance of the treated populations, and management of the patients who develop a SAE. Though lower, the risk that SAE occur in areas where the prevalence of Loa microfilaraemia is lower than 20% is not nil, and thus guidelines have also been developed regarding the strategy to apply in such situations [This paper defines a model which identifies areas of potential high risk of severe adverse reactions to ivermectin, and will contribute to APOC's programme development by enabling resources to be effectively targeted to areas deemed at risk. The Technical Consultative Committee of APOC, and the Mectizan Expert Committee have developed a series of recommendations aimed at "facilitating effective detection and management of SAEs following treatment with Mectizan in known and suspected tuations .Loa loa. As it stands, the model presented here uses environmental features known to be associated with the biology of the vector, is robust for the area of data collection and predicts areas within Cameroon where Loa loa has been found in the past. It provides a rapid assessment methodology of areas where adverse reactions to ivermectin may occur and will be further developed in conjunction with the RAPLOA procedure which APOC intends to apply to CDTI areas potentially endemic for Loa loa [The model developed is by no means a definitive product but provides a basis for decision making in terms of where rapid epidemiological surveys for loiasis should now be targeted. Modelling in this way permits an iterative process between field epidemiologist and modeller which not only means that decisions are made on the best available data but that such data is updated rapidly as new survey results are entered in the database and the model refined. Models are valuable in so far as they reflect reality. Given the complexity of disease transmission processes where human, parasite, vector and environment interact, it is impossible to think that all relevant factors can be incorporated into a general model which can be applied on a regional scale. What is significant here is the fact that important decisions need to be made now with regard to the likely spatial extent of the distribution of  Loa loa . The nexAPOC African Programme for Onchocerciasis ControlCDTI Community-Directed Treatment with IvermectinCPC Centre Pasteur du CamerounDEM Digital Elevation ModelLoa loaERM Environmental Risk Map for GIS Geographic Information SystemGPELF Global Programme to Eliminate Lymphatic FilariasisIRD Institut de Recherche pour le DéveloppementMfs MicrofilariaeNDVI Normalised Difference Vegetation IndexNOAA-AVHRR National Ocean and Atmospheric Administration – Advanced Very High Resolution RadiometerRAPLOA Rapid Assessment of the Prevalence and Intensity of Loa infectionSAE Serious Adverse EventSPOT Satellite Pour l'Observation de la TerreTDR UNICEF/UNDP/World Bank/WHO Special Programme for Research and Training in Tropical DiseasesUNEP-GRID United Nations Environment Programme-Global Resource Information DatabaseUSGS United States Geological SurveyNone declared.MCT designed the study, planned the analysis, supervised the modelling, interpreted the results, and wrote the paper.VO prepared the data, developed the model, prepared the maps, and wrote the paper.JK and JG collected the field data used for the development of the model.SW, IT and PE collected the field data used for the validation of the model.JHR supervised the study thanks to which the validation data were collected.DHM proposed the study, contributed to its design and interpretation of the results.MB designed the study, planned the analysis, collected the field data, interpreted the results, and wrote the paper.


These mutant embryos exhibit U-shaped somites characteristic of defects in slow muscle development. In addition, Hedgehog pathway mutations disrupt spinal cord patterning. We report the positional cloning of you, one of the original you-class mutations, and show that it is required for Hedgehog signaling in the development of slow muscle and in the specification of ventral fates in the spinal cord. The you gene encodes a novel protein with conserved EGF and CUB domains and a secretory pathway signal sequence. Epistasis experiments support an extracellular role for You upstream of the Hedgehog response mechanism. Analysis of chimeras indicates that you mutant cells can appropriately respond to Hedgehog signaling in a wild-type environment. Additional chimera analysis indicates that wild-type you gene function is not required in axial Hedgehog-producing cells, suggesting that You is essential for transport or stability of Hedgehog signals in the extracellular environment. Our positional cloning and functional studies demonstrate that You is a novel extracellular component of the Hedgehog pathway in vertebrates.Hedgehog signaling is required for many aspects of development in vertebrates and invertebrates. Misregulation of the Hedgehog pathway causes developmental abnormalities and has been implicated in certain types of cancer. Large-scale genetic screens in zebrafish have identified a group of mutations, termed  Genetic studies in zebrafish have identified a new protein involved in Hedgehog signaling - a key pathway required for development in vertebrates and invertebrates Drosophila larval segments and imaginal discs, dorsoventral patterning of the vertebrate neural tube, and anterior–posterior patterning of vertebrate limbs; in addition, many recent studies have illuminated the widespread and conserved role of Hedgehog signaling in development . Misregiewed in ,3,4, mediewed in ,6,7, paniewed in , and holiewed in ,10.Drosophila, diffusion of lipid-modified Hedgehog proteins is dependent on the action of tout velu, a gene involved in the synthesis of heparan sulfate proteoglycans [growth-arrest specific gene product Gas1 [After release from signaling cells, the activity and distribution of Hedgehog proteins are modulated by a variety of factors in the extracellular environment. In oglycans ,12. The oglycans . In vertuct Gas1 , and Heduct Gas1 . Moreoveuct Gas1 ,17. Geneuct Gas1 ,19.prox1. In contrast, fast muscle fibers can be identified via their multinucleate morphology and lack of prox1 expression [Hedgehog pathway function in zebrafish has been analyzed primarily in the context of skeletal muscle development and differentiation ,26,27,28pression . As devepression ,27,28. Spression ,28,29,30pression ,28.Many lines of evidence indicate that Hedgehog signals from axial tissues specify slow muscle in zebrafish. Slow muscle fibers are reduced or absent in embryos with Hedgehog pathway mutations ,31,32,33you-class” mutants because of their U-shaped somites. Five of the seven you-class mutations have been cloned, and four of these genes, syu/shh, yot/gli2, smu/smoh, and con/disp1, encode members of the Hedgehog signaling pathway [ubo/prdm1, which encodes a transcriptional switch that acts downstream of Hedgehog signaling in the development of slow muscle [ubo and Hedgehog pathway mutant phenotypes. For example, Hedgehog pathway mutants have defects in the lateral floor plate of the neural tube and the dorsal aorta, which are apparently normal in ubo mutants [ubo: Hedgehog pathway mutations reduce or abolish expression of the Hedgehog target ptc1, whereas ptc1 expression is normal in ubo mutants, indicating that they can receive Hedgehog signals [Genetic screens have identified a number of mutations disrupting the Hedgehog pathway in zebrafish ,39,40,41 pathway ,36,37,39w muscle ,42. Care mutants ,43. Exam signals ,33,36,39you-class gene, you, has revealed delayed development of the dorsal aorta and the absence of lateral floor plate marker expression in addition to slow muscle defects [ptc1 and adaxial myod, is reduced in you mutants [you gene acts within the Hedgehog pathway itself rather than downstream of Hedgehog signaling in processes specific to slow muscle development. Prior to this study, the molecular identity of the you gene has remained unknown. We report the positional cloning of the you gene and show that it encodes a novel extracellular EGF-CUB  protein required for Hedgehog signaling. Functional studies provide evidence that you is essential for the transport or stability of Hedgehog signals in the extracellular environment.Previous phenotypic characterization of mutants for the eponymous  defects ,43. More mutants . These ryou mutants lacked the horizontal myoseptum and exhibit the U-shaped morphology that defines mutants of the you class . These hedgehog genes—ehh, shh, and twhh—are expressed at the midline in early embryonic stages. To determine whether you is required for hedgehog gene transcription, we analyzed the expression of hedgehog genes in wild-type and you mutant embryos. Expression of all three hedgehog genes appeared normal in you embryos at bud stage  region of LG 7, between simple sequence length polymorphism (SSLP) markers Z11119 and Z15270  library and initiated a chromosome walk beginning with BAC zC172H20. After identifying polymorphisms in BAC end sequences, testing these polymorphisms on our mapping panel, and iteratively rescreening the pooled BAC library, we identified a contiguous stretch of genomic sequence spanning portions of five BACs with ends that flanked you  and SCUBE2 in human (66% identity). The orthology of these genes was further supported by comparative mapping: the human genes SCUBE2, LMO1, STK33, and ST5 exhibited conserved synteny with orthologous genes in both mouse and zebrafish . Pairwibrafish , the C-terminal sequence following the CUB domain (90% identity), and the EGF repeats (74% identity). A spacer region in the center of the amino acid sequence showed lower conservation (47% identity). Examination of this spacer region in the vicinity of the CUB domain revealed a repeated motif of six cysteine residues with characteristic and regular spacing, shown in yellow in SCUBE proteins are characterized by a signal peptide and by two types of conserved extracellular domains: EGF and CUB . In all ty97you mutants, a C to T transition alters the coding sequence at residue 644, changing a glutamine codon to a stop codon (In op codon A and 3B.op codon C.you mutants, we performed morpholino oligonucleotide (MO) injection experiments to phenocopy defects seen in you, and mRNA injection experiments to rescue the you phenotype in mutants (myod (n = 112) in the adaxial cells (n = 70) in the muscle pioneers (myod (n = 65) or Engrailed (n = 48) expression  of embryos from you/+ intercrosses showed expression of myod in adaxial cells at 12 somites (myod expression from these intercrosses showed that 137 (24%) were homozygous mutant for you. In contrast, 23.6% (n = 127) of embryos from you/+ intercrosses injected with a mutant form of you mRNA lacked expression of myod in adaxial cells  from a you/+ intercross injected with wild-type mRNA showed strong Engrailed labeling in the muscle pioneers (ty97you), whereas 24% (n = 33) of embryos injected with control mRNA lacked Engrailed expression (you mutation). In addition, injection of you MOs resulted in loss of nkx2.2 expression in the trunk and tail of wild-type embryos at 24 hpf, and injection of 50 pg of you mRNA was sufficient to rescue nkx2.2 expression in you mutants (data not shown).When injected with 50 pg of synthetic wild-type  somites E. Genotyal cells F; 20 of you transcripts appear to be maternally deposited in zebrafish embryos (you transcripts in the embryo began to be restricted, and at bud stage (10 hpf) you was expressed in the eye field, in distinct bilateral domains within the developing brain, and in the developing trunk of the embryo in broad paraxial stripes (you expression continued to be refined, such that six-somite embryos exhibited expression in the eye field, in stripes in the midbrain and the midbrain–hindbrain boundary (MHB), in a complex pattern in the hindbrain, and in paraxial stripes along the anterior–posterior axis  of synthetic you mRNA that was sufficient to rescue the phenotypic defects observed in you mutants  was injected into embryos from a you/+ intercross, ectopic expression of myod . Because downstream targets of the Hedgehog pathway were rescued or upregulated in shh-injected you mutants, components of the Hedgehog pathway downstream of shh are most likely functional in you embryos. These results are consistent with you acting upstream of or parallel with shh in the Hedgehog pathway.To explore the possibility that the  mutants . When con = 394; B, Engrai n = 25; F, or nkx n = 17; J. This r n = 53; C and 6D, n = 78; K and 6L patched activity with MOs  or a combination of MOs targeting both ptc1 and ptc2 (n = 13 mutants). you mutant embryos injected with a ptc1 mismatch control MO did not exhibit rescued myod expression (n = 8 mutants). In similar experiments, injection of patched MOs was sufficient to rescue or expand Engrailed expression in muscle pioneers of you mutant embryos .Additional evidence that You acts upstream of the Hedgehog response derived from a loss-of-function approach, in which we activated the Hedgehog pathway by knocking down with MOs . Expressyou function to respond to Hedgehog signaling, we created genetic chimeras by transplanting cells from mutant embryos into wild-type hosts (you mutant embryos were able to differentiate as muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei of mononucleate cells at the proper position in the somite (n = 13 embryos). Similarly, cells from embryos in which you function had been reduced with MOs were able to differentiate as Engrailed-expressing muscle pioneers when introduced into embryos treated with mismatch control MOs (data not shown).To determine whether a cell must be wild-type for pe hosts . Cells dyou function in order for target cells to appropriately respond to Hedgehog signals, we transplanted cells derived from wild-type donors into you mutant hosts. Of 91 chimeric mutant hosts, ten embryos exhibited rescue of Engrailed expression in genotypically mutant muscle pioneers, as defined by characteristic strong Engrailed expression in elongate nuclei at the proper position of posterior somites, where Engrailed is not normally expressed in you mutants .you is essential for Hedgehog signaling, our analysis indicates that you mutant cells are able to produce and respond to Hedgehog signals. hedgehog gene expression in the embryonic midline is normal in you mutants, indicating that You acts downstream of hedgehog gene transcription  in wild-type embryos did not result in obvious phenotypes when assayed by gross morphology or by expression of Hedgehog target genes, including adaxial myod, Engrailed in muscle pioneers, or nkx2.2 in the ventral spinal cord . For initial localization of you, bulked segregant analysis  was perescribed .http://www.rzpd.de). BAC end sequences were obtained from the Sanger Institute database  following double digest with either Pst I and EcoR I or Xba I and Xho I. Sequences were analyzed on a 3730 DNA Analyzer . Sequences generated from this BAC were used in iterated searches against the zebrafish whole-genome shotgun assembly to nucleate contigs of genomic sequence. Sequencing primers were designed from these contigs and used to generate additional sequence data for zC93A15.The CHORI211 BAC library was screened by PCR to identify positive BAC clones (you clone in pBluescript SK− (Stratagene) was isolated from a 15–19-hpf cDNA library (gift of Bruce Appel and Judith Eisen). A cDNA clone harboring a frameshift mutation that is predicted to truncate the you protein at amino acid residue 34 was isolated from the same library and was used as a control in overexpression experiments. A modified version of the pCS2+ expression vector was generated by cloning a 41-bp fragment into its EcoR I and Xba I sites. This stuffer fragment abolished the endogenous EcoR I, Stu I, Xho I, and Xba I restriction digest sites of pCS2+, and introduced Xba I, Sac I, Apa I, Pst I, and Xho I recognition sequences in a 5′ to 3′ orientation with respect to the SP6 promoter. Wild-type and mutant you clones were subcloned into the Xba I and Xho I sites of this modified pCS2+ vector. In situ probes for you were generated by linearizing this vector with Xba I, followed by antisense RNA synthesis with T3 polymerase. Synthetic you mRNA for injections was generated by digestion with Not I, followed by transcription using the SP6 mMessage mMachine kit (Ambion, Austin, Texas, United States).A full-length you/+ intercrosses were genotyped after in situ hybridization and antibody labeling as described [myod [ptc1 [nkx2.2 [ehh [shh [Probe synthesis, in situ hybridizations, and immunohistochemistry were performed using standard protocols. Embryos from escribed . Other ped [myod , ptc1 [6od [ptc1 , nkx2.2  [nkx2.2 , ehh [20ehh [shh , and monehh [shh . Genotypyou translational initiation site (5′-GCCGTACAGTCCAAACAGCTCCCAT-3′) or a 5-bp mismatch control MO (5′-GCCcTAgAGTCgAAACAcCTgCCAT-3′) was diluted in a 1x Danieau's solution with Phenol Red at 5 mg/ml prior to microinjection. Sequences for MOs targeting ptc1 and ptc2 were obtained from [Embryos were injected through their chorions with 500 pl of solution at the 1–4-cell stage as described . RNA wasned from .you/+ intercrosses were injected at the 1–4-cell stage with a 1% solution of Oregon Green 488 dextran (Molecular Probes, Eugene, Oregon, United States). Approximately 50–100 cells were removed from labeled donors in late blastula and early gastrula periods (4–5.3 hpf) and transplanted near the margin of unlabeled sibling hosts. Labeled donor embryos were allowed to develop until 24 hpf. Genotypes of donor embryos derived from you/+ intercrosses were determined by PCR. Genotypes of host embryos were determined by staining with Engrailed antibody. Donor cells in chimeras with wild-type cells transplanted into you mutant hosts were obtained either from WIK intercrosses or from genotypically wild-type embryos in you/+ intercrosses.Cellular transplantations were done according to standard methods . Embryosyou cDNA sequence has been deposited in GenBank (http://www.ncbi.nlm.nih.gov/Genbank/index.html) under accession number AY741664.The http://www.ncbi.nlm.nih.gov/LocusLink/) accession numbers for the genes and gene products discussed in this paper are Attractin (LocusID 8455), C1r (LocusID 715), C1s (LocusID 716), con/disp1 (LocusID 378448), Cubilin (LocusID 8029), ehh (LocusID 30299), Engrailed (LocusID 30244), Gas1 (LocusID 14451), Hedgehog (LocusID 42737), Hip1 (LocusID 15245), Megalin (LocusID 14725), myod (LocusID 30513), nkx2.2 (LocusID 30697), Patched (LocusID 35851), prox1 (LocusID 30679), ptc1 (LocusID 30181), ptc2 (LocusID 30189), sea urchin Fibropellin (LocusID 373313), smu/smoh (LocusID 30225), syu/ssh (LocusID 30269), Tolloid (LocusID 42945), tout velu (LocusID 36614), twhh (LocusID 30444), ubo/prdm1 (LocusID 323473), yot/gli2 (LocusID 30154), human LMO1 (LocusID 4004), human SCUBE1 (LocusID 80274), human SCUBE2 (LocusID 57758), human ST5 (LocusID 6764), human STK33 (LocusID 65975), mouse SCUBE1 (LocusID 64706), and mouse SCUBE2 (LocusID 56788).The LocusLink (


Harpagophytum procumbens preparations in the treatment of various forms of musculoskeletal pain.The objective of this review is to determine the effectiveness of Harpagophytum preparations in adults suffering from pain due to osteoarthritis or low back pain.Several databases and other sources were searched to identify randomized controlled trials, quasi-randomized controlled trials, and controlled clinical trials testing Given the clinical heterogeneity and insufficient data for statistical pooling, trials were described in a narrative way, taking into consideration methodological quality scores. Twelve trials were included with six investigating osteoarthritis , four low back pain, and three mixed-pain conditions.Harpagophytum extract containing less than <30 mg harpagoside per day in the treatment of knee and hip osteoarthritis. There is moderate evidence of effectiveness for (1) the use of a Harpagophytum powder at 60 mg harpagoside in the treatment of osteoarthritis of the spine, hip and knee; (2) the use of an aqueous Harpagophytum extract at a daily dose of 100 mg harpagoside in the treatment of acute exacerbations of chronic non-specific low back pain; and (3) the use of an aqueous extract of Harpagophytum procumbens at 60 mg harpagoside being non-inferior to 12.5 mg rofecoxib per day for chronic non-specific low-back pain (NSLBP) in the short term. Strong evidence exists for the use of an aqueous Harpagophytum extract at a daily dose equivalent of 50 mg harpagoside in the treatment of acute exacerbations of chronic NSLBP.There is limited evidence for an ethanolic  Harpagophytum procumbens (H) for the treatment of various diseases, including musculoskeletal complaints. For more than half a century, various preparations from H have been continuously used in Europe and have become an established traditional treatment for rheumatic complaints. The monograph of the European Scientific Cooperative on Phytotherapy (ESCOP) [Natives in the steppes of South and Southwest Africa use the secondary root tubers of  (ESCOP)  recommen (ESCOP)  the extr (ESCOP) .Recent in-vitro studies indicate that preparations from H may interact with the inflammatory cascade, including the cytokines -6. MoreoHarpagophytum preparations in the treatment of musculoskeletal pain.The objective of this review was to determine the effectiveness of H. procumbens) and musculoskeletal conditions. Search strategies were modified for each database [for PUBMED strategy see appendix 2]. One reviewer (SC) contacted experts and acquired relevant citations. In addition, manufacturers of commercial Harpagophytum preparations and content experts were contacted and asked to contribute published and unpublished material. Reference lists in review articles and the retrieved trials were searched for further trials.Two reviewers (JG) and (SC) conducted electronic searches using the following databases: PUBMED (1966 up to September 16, 2003), EMBASE (OVID technologies: 1980 to wk 40 2003), Cochrane Controlled Trials Registry, Cochrane Musculoskeletal specialized register, Dissertation Abstracts, BIDS ISI, and the Cochrane Complementary Medicine Fields Specialized Register. The search strategy was developed by combining a highly sensitive method for isolating controlled clinical trials developed for the Cochrane Collaboration [Trials that met the criteria outlined in Table Two reviewers (SC, JG) extracted data from each trial using a standardized form. The following data were extracted from each study: authors' names; date of publication; country of origin; type of study, including number of study centers; participants (numbers, disease(s), characteristics of the study population (age, size, weight, gender)); duration of acute exacerbation or chronic disease; baseline values with details on pain and previous treatments; additional treatments; types of outcome measures; summary statistics; timing of outcome assessment; withdrawals and drop-outs; and adverse events. Blinding to authors, institution or journal title was not done, given the contradictory evidence relating blinded reviewing and bias ; additioMethodological quality was assessed using the criteria list developed by van Tulder et al  ,11. SpecHigh quality studies are defined as those that fulfill more than 50% (>6) of the quality criteria. Sensitivity analyses were carried out to explore the results when the definition of high quality trials was set at 40 % (>5) and 60% (>7) fulfillment of the quality criteria.According to the Van Tulder Scoring ,11, the Strong – consistent findings among multiple high quality RCTs1. Moderate – consistent findings among multiple low quality RCTs and/or CCTs and/or one high quality RCT2. Limited – one low quality RCT and/or CCT3. Conflicting – inconsistent findings among multiple trials (RCTs and/or CCTs)4. No evidence from trials – no RCTs or CCTs5. Planned subgroup analyses included: (1) pain site, (2) type of pain (acute (≤ 6 weeks duration), sub-acute (6 to 12 weeks duration), and chronic pain (> 12 weeks)), and (3) comparison  versus placebo and botanical medicine versus other treatment).A total of 130 citations were isolated from electronic searches and all abstracts were retrieved. A total of three additional references were supplied by content experts. A total of 120 papers were excluded because of publication type (reviews or reports) or because of improper trial design. The complete references were retrieved for the remaining 13 trials. All 13 references were included in the current review . Of thesA total of five randomized trials included 385 patients (range 46 to 122) with osteoarthritis of the hip or the knee ,18,21-23A total of four trials included 505 patients (range 88 to 197) with acute exacerbation's of chronic non-specific low back pain -17. All The last three trials included 215 patients with various forms of musculoskeletal pain (range 50 to 100) ,20,24. SAll trials, except for two ,24, repoRatings for each trial on each quality criterion are reported in Table Of the trials utilizing subjects with acute exacerbations of chronic non-specific low back pain, all four were considered to be of high methodological quality regardless of the cutoff -17. MethOf those trials including a mixed sample of subjects ,20,24, tLecomte & Costa (1992)  utilizedR) or Diacerhein (D), to 122 subjects with acute exacerbations of hip and knee. Groups did not differ significantly in sponanteous pain or the Lequesne index, though differences from baseline were larger for the H group. Subjects in the H group used less diclofenac (mean = 21 tablets) than those in the D group (60 tablets) and also used less acetominophen-caffeine (H = 40 tablets and D = 60 tablets).Chantre et al (2000)  gave H cR or placebo in addition to ibuprofen (at 800 mg ibuprofen during weeks 1–8, 400 mg during weeks 9 – 16), and only H or placebo during weeks 17–20. The main outcome measure was the responder rate, which allowed a WOMAC pain score increase of up to 20% and no additional consumption of ibuprofen in weeks 17 to 20. Results favoured the H group.Biller (2002)  gave kneR or placebo in addition to ibuprofen (at 800 mg ibuprofen during weeks 1–8, 400 mg during weeks 9–16) and only H or placebo during weeks 17–20. The main outcome was the responder rate, which was defined as the number of patients that required fewer than 4000 mg ibuprofen and had a pain score increase of no more than 20% on the WOMAC component pain during weeks 17 to 20. Results indicated more responders in the H group.Frerick et al (2001)  gave 46 R,) with phenylbutazone among 40 individuals with acute exacerbations of rheumatic joint and muscle pain and 10 with gouty arthritis. Results favoured the H group.Schruffler (1980)  comparedR,, or placebo. Results favoured the H group (see Table 4). Of the 59 patients in the H group, five dropped out, one of these due to tachycardia. Of the 59 patients in the P group, four dropped out for unknown reasons. A total of four adverse effects occurred in the H group. These consisted of two individuals with nausea/emesis due to the tramadol, one patient with repeated tussive irritation, and the patient with tachycardia mentioned above. A total of 10 adverse events occurred in the P group. These included nausea (N = 2), and one patient each of nausea/vertigo due to tramadol, fatigue/vertigo, vertigo alone, diuresis/normalization of constipation (i.e. intractable constipation), constipation , diuresis , and sleep disturbances (permanent).In the Chrubasik study (1996) , 128 pat50) or 9000 mg H /day (with 100 mg harpagoside per day, H100), or placebo . Participants had acute exacerbations of non-specific low back pain; current pain that was > 5 on a VAS (0–10). The median durations of chronic pain were P 15 years, H50 15 years, H100 15 years. The number of patients with acute exacerbations of greater than 3 months for each group was P 54 (82%), H50 53 (82%), H100 55 (83%). Current pain and Arhus scores were similar among all groups. The principle outcome measure, the number of patients who were pain-free without the permitted rescue medication for 5 days out of the last treatment week was 3 (P), 6 (H50) and 10 (H100) (p = 0.027, one-tailed Cochrane-Armitage test). The authors found significant improvements in pain in both H groups as compared with the placebo group. A subgroup analysis found differences between groups for those without pain radiating to the legs  and for those without a neurological deficit .An additional study by Chrubasik et al (1999)  randomizR (N = 44) or VioxxR . The percentage of subjects with chronic pain of > 6 days for each group was H 84%, R 84% and acute pain of > 90 days in H 91%, R 89%. Results indicated statistically non-significant difference between H and R. Also, a total of 21 (group H) and 13 (group R) patients used tramadol, with the average consumption being 230 mg (H) and 133 mg (R). A total of seven drop-outs occurred due to adverse events ; two others resulted from excessive low back pain (R = 2). There were a total of seven protocol violations. A total of 14 participants in each group had adverse events with gastrointenstinal complaints, equaling eight in the H group and nine in the R group, with more severe events in the R group. Two adverse events were reported to be unrelated to the study medication in the H group.Chrubasik et al (2003)  randomizR or conventional treatments (NSAIDs, exercise, massage, nerve blocks, acupuncture). Subjects' pain was > 5 (VAS 0–10) on at least two of the following five scores: pain at rest, while sitting, lying, and walking or at night. The median duration of acute pain was six weeks for both groups. The median duration of chronic pain was 120 months for the H group and 72 months for the control group. There were no statistically significant differences between the groups.In another study by Chrubasik et al (1997) , particiIn a placebo controlled double-blind study, Guyader (1984)  includedR containing extract based on 4500 mg crude plant material  per day or a placebo for 30 days. Outcome measures included a subjective pain scale (no pain, mild pain, moderate pain, severe pain, excrutiating pain) at baseline and after four weeks of treatment. The number of pain-free individuals with low back pain was H n = 4, P n = 2, and for other pain sites H n = 5, P n = 0.In a double-blind, placebo-controlled trial, Schmelz et al (1997)  randomizGobel et al (2001)  conducteOsteoarthritis of the knee, hip, and spine as well as non-specific low back pain may be associated with pain, stiffness, limitation of function, and diminished quality of life . AlthougHarpagophytum procumbens may be effective in various types of musculoskeletal pain conditions. Statistical pooling was not possible because of a lack of adequate data and clinical heterogeneity. The sensitivity analysis for methodological quality revealed that the trials on low back pain were of high quality, the trials on osteoarthritis were of high quality except one moderate quality study, and the trials on mixed pain conditions were of moderate quality. The quality of reporting in most of these trials was good. In order to increase transparency, trialists should refer to the CONSORT statement in designing and reporting clinical trials of herbal medicinal products [This qualitative analysis of the 12 trials suggests that specific preparations and doses of products .One high quality trial indicates that there is moderate evidence of effectiveness for powdered H plant material at a dose equivalent to 60 mg of harpagoside per day for osteoarthritis of the spine, hip, and knee. However, because of the clinical heterogeneity of patients in this trial, a confirmatory study is required to firmly establish efficacy for each location of osteoarthritis. In one high quality study, 4500 mg powder containing 57 mg harpagoside in the daily dosage showed moderate evidence for non-inferiority to diacerhein in patients suffering from acute exacerbations of osteoarthritis in the hip and knee.Harpagophytum procumbens in osteoathritis. These trials must include homogenous pain conditions and must test H against standard osteoarthitis medications. Additionally, trialists should consider using symptom severity outcome measures that have proven validity and reliability, such as visual analogue scales [Two trials employed an ethanolic extract (solvent 60% ethanol) containing less than 30 mg harpagoside per day in patients with osteoarthritis of the knee  and hip e scales -37, ostee scales -38, and e scales ,37,38.A total of four high quality trials tested various dosages of Hextract in acute exacerbations of chronic non-specific low back pain. Two trials with a total of 325 patients showed that an aqueous extract at the equivalent daily dosage of 50 mg harpagoside appears to reduce pain in patients with acute episodes of chronic NSLBP greater than does placebo ,16. TherHarpagophytum procumbens against standard treatments will clarify relative efficacy and safety.An aqueous extract of H at the equivalent daily dose of 60 mg harpagoside appears to be equivalent to 12.5 mg Rofecoxib in improving pain in individuals with acute episodes of chronicNSLBP . TherefoThe final trials  the use of a Harpagophytum powder at 60 mg harpagoside in the treatment of osteoarthritis of the spine, hip and knee; (2) the use of an aqueous Harpagophytum extract at a daily dose of 100 mg harpagoside in the treatment of acute exacerbations of chronic non-specific low back pain; and (3) the use of an aqueous extract of Harpagophytum procumbens at 60 mg harpagoside being non-inferior to 12.5 mg rofecoxib per day for chronic non-specific low back pain (NSLBP) in the short term. Strong evidence exists for the use of an aqueous Harpagophytum extract at a daily dose equivalent of 50 mg harpagoside in the treatment of acute exacerbations of chronic NSLBP.There is limited evidence for an ethanolic One individual (SC) was an author of several original trials included in this systematic review. This did not appear to influence the content of this paper.JG developed the initial idea for the manuscript, searched for trials, extracted data and wrote and edited the manuscript.SC developed the initial idea for the manuscript, searched for trials, extracted data, and wrote and edited the manuscript.EM extracted data and wrote and edited the manuscript.Highly sensitive search strategy for randomized controlled trial searches using PUBMED OR ((singl* [tw] OR doubl* [tw] OR trebl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* {tw])) OR (latin square [tw]) OR placebos [mh] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR comparative study [mh] OR evaluation studies [mh] OR follow-up studies [mh] OR prospective studies [mh] OR cross-over studies [mh] OR control* [tw] OR prospectiv* [tw] or volunteer* [tw]) NOT PUBMED search strategy OR ((singl* [tw] OR doubl* [tw] OR trebl* [tw] OR tripl* [tw]) AND (mask* [tw] OR blind* {tw])) OR (latin square [tw]) OR placebos [mh] OR placebo* [tw] OR random* [tw] OR research design [mh:noexp] OR comparative study [mh] OR evaluation studies [mh] OR follow-up studies [mh] OR prospective studies [mh] OR cross-over studies [mh] OR control* [tw] OR prospectiv* [tw] or volunteer* [tw]) NOT  AND ("harpagophytum procumbens" OR (devil's AND claw)) AND  NOT reviewThe pre-publication history for this paper can be accessed here:


Disorders of coagulation have long been associated with inflammatory bowel disease. Children, as well as adults, with both active and inactive ulcerative colitis have been found to have abnormal coagulation and fibrinolysis. Disseminated intravascular coagulation arises from an overwhelming of the haemostatic regulatory mechanisms leading to an excessive generation of thrombin and a failure of the normal inhibitory pathways to prevent systemic effects of this enzyme. Ulcerative colitis has been associated with disseminated intravascular coagulation in conjunction with septicemia, toxic megacolon and surgery.A fourteen-year-old boy with a history of poorly controlled ulcerative colitis presented with nonbilious emesis, hematochezia, and hematuria. Laboratory workup revealed disseminated intravascular coagulation. He was placed on triple antibiotics therapy. An infectious workup came back negative. A computerized tomography (CT) scan of the abdomen revealed a marked thickening and irregularity of the bladder wall as well as wall thickening of the rectosigmoid, ascending, transverse, and descending colon. Patient's clinical status remained stable despite a worsening of laboratory values associated with disseminated intravascular coagulation. Patient was begun on high dose intravenous steroids with improvement of the disseminated intravascular coagulation laboratory values within 12 hours and resolution of disseminated intravascular coagulopathy within 4 days. A thorough infectious workup revealed no other causes to his disseminated intravascular coagulation.The spectrum of hypercoagulable states associated with ulcerative colitis varies from mild to severe. Although disseminated intravascular coagulation associated with ulcerative colitis is usually related to septicemia, toxic megacolon or surgery, we present a case of an ulcerative colitis flare resulting in disseminated intravascular coagulation and a bladder hematoma. A wide variety of disorders are associated with the development of disseminated intravascular coagulation (DIC). Initiation usually involves mechanical tissue injury and or endothelial cell activation and injury. DIC arises from an overwhelming of the haemostatic regulatory mechanisms leading to an excessive generation of thrombin and a failure of the normal inhibitory pathways to prevent systemic effects of the enzyme leading to DIC . UlceratA 14-year-old boy with a diagnosis of ulcerative colitis based on colonic histology, serology and a normal barium study of his small bowels was admitted with a five-day history of nonbilious vomiting and bloody diarrhea. Additional symptoms included recent onset hematuria, and low-grade fevers to 100.4 C over the prior four days. He had also sustained a 25 lb weight loss in the last six months, indicating a lack of disease control.As an outpatient, his maintenance therapy included mesalamine (1 gram three times a day), and mercaptopurine (75 mg once per day). In addition, he had been started on prednisone approximately 7 weeks prior for treatment of an ulcerative colitis flare. His current dose of prednisone was 10 mg once a day. Soon after symptoms begun, he had been placed on ciprofloxacin as treatment for a presumptive flare.Physical exam showed he was afebrile, with a heart rate of 130 beats per minute, respiratory 16 breaths per minute and blood pressure 115/67 mmHg. He was alert although with a sallow appearance. Abdominal exam revealed a soft nontender nondistended abdomen. Rectal showed normal external exam with grossly bloody stool. Initial blood work showed hemoglobin of 12.3, a normal white blood cell count, normal differential and normal platelet count with a mildly elevated prothrombin time of 16.2 with an international normalized ratio (INR) of 1.2. Urine analysis showed a specific gravity of 1.035, 3+blood, +ketones and > 100 RBC per high powered field and 0–5 WBC per high power field. Abdominal ultrasound revealed irregular shaped bladder wall.Clostridium difficile toxin analysis.Patient was placed on intravenous fluids (IV) as well as metronidazole (IV). Blood and urine cultures were sent for analysis. Stool was sent for culture and for 3) increasing PT/PTT (21.3/47 seconds) with an INR of 1.8. Blood smear showed moderate amount of elliptocytes, schistocytes, microcytes and fragmented red blood cells. Initial DIC panel revealed an elevated D-dimer of 4.9 mcg/mL with a normal thrombin time and fibrinogen. Thrombin time subsequently increased to > 120 seconds. D-dimers increased to 10.3 mcg/mL. A computerized tomography (CT) scan of the abdomen revealed a marked thickening and irregularity of the bladder wall as well as wall thickening of the rectosigmoid, ascending, transverse, and descending colon  and platelet count (64 K/mmPatient was begun on broad-spectrum antibiotics because of concerns regarding possible bacteremia and a worsening DIC laboratory picture. Blood, stool and urine cultures returned negative. Viral cultures and monoclonal antibody staining for adenovirus detection in the urine was negative. Despite a worsening in the DIC panel, the patient remained clinically unchanged. IV steroids were begun approximately 36 hours into patient's hospital stay. Patient had a stabilization of PT/PTT/INR/thrombin time and D-dimer, and a subsequent normalization of labs over the following 4-day period  Figure , 8 . PatDisorders of coagulation have long been associated with inflammatory bowel disease -11. ChilAlthough hypocoagulable states have been noted in the literature, most studies indicate an associated hypercoagulable state. There appears to be an increase in thrombin-anti-thrombin complex and a decrease in antithrombin III activity, which causes an increase in thrombin generation,12,13. OThe hypercoagulable state in ulcerative colitis is associated thromboembolic events; although uncommon, deep vein thrombosis, pulmonary embolisms and stroke have been associated with ulcerative colitis,15-18. DPresented is a case of DIC associated solely with an ulcerative colitis flare resulting in a bladder hematoma. We presume that the occurrence of DIC in this patient resulted from an acute flare on top of a chronic unremitting course of ulcerative colitis. A thorough infectious work-up of this patient did not reveal any infectious etiology that would have predisposed him to develop DIC. The presumed cause of the DIC was damage to the endothelial wall of the colonic blood vessels, which exposed blood to excessive amounts of tissue factor. This in turn led to the excessive generation of thrombin and a failure of the normal coagulation inhibitory pathways. By treating the ulcerative colitis flare, we decreased the intestinal inflammation and thereby decreased the endothelial cell damage. This, theoretically, resolved the DIC. Patient's clinical symptoms and laboratory values normalized after treatment with intravenous steroids, completely resolving the disseminated intravascular coagulopathy.The authors declare that they have no competing interests.DLS drafted the manuscript. KM and DC participated in the manuscript preparation. All authors approved the final manuscript.The pre-publication history for this paper can be accessed here:


It is believed that the development of gastric cancer (GC) before the age of 50 has a hereditary basis. Blood group A and history of gastric cancer in first-degree relatives have been shown to be risk factors for GC.In this case-control study, we enrolled patients with GC who were diagnosed before the age of 50. Patients who were diagnosed as having GC were selected. A total of 534 cases were found; of these, 44 diagnosed before the age of 50 were included in the case group. For the control group, 22 males and 22 females were randomly selected from the remaining subjects, who had diagnoses of GC after the age of 50. All the surviving patients and family members of the dead patients were interviewed about the history of cancer in the family and the age at which other family members developed cancer. The blood group of each subject was also obtained.forty-four cases under 50 years old (mean age: 36.2 years) and forty-four controls (mean age: 67.1 years) were enrolled in the study. At the time of the study, 59.1% of the study group and 50% of the control group were alive . In the study group, 68.1%, 13.6%, 13.6% and 4.5% had blood groups O, A, B and AB, respectively. In the control group the corresponding figures were 27.7%, 63.6%, 6.8% and 4.5%. First or second-degree relatives with cancer, including gastric (the most frequent), breast, lung, gynecological and hematological malignancies, were noted in 54.5% of the cases and 11.4% of the controls (p < 0.01). Family histories of cancer were accepted as valid provided that they were based on valid medical documents.It seems that the development of GC before the age of 50 is likely to be accompanied by familial susceptibility. Interestingly, our study showed a significant correlation between blood group O and the development of gastric cancer under the age of 50. Gastric cancer is the second most common cause of cancer-related death in the world . Its incThe reduced incidence of gastric cancer in western countries reflects a decrease in cancers arising in the distal stomach (body and antrum). In contrast, the incidence of cancer in the proximal stomach and esophagogastric junction has steadily increased, at a rate exceeding that of any other cancer except melanoma and lung cancer -13. In aIn general, familial genetic mechanisms do not play as important a role in gastric cancer as they do in e.g. colorectal cancer. Nonetheless, in some regions, a family history of gastric cancer may be a risk factor for the disease, although this might reflect environmental factors shared by members of a family . Rate coIn a review about genetic predisposition to gastric cancer, Bevan and Houlston 1999) concluded that several genes may be associated with increased risk [99 concluMany studies have addressed the correlation between ABO antigens and the development of gastric cancer, but most of these have indicated a correlation between sporadic cases of gastric cancer and blood group A. This association further supports the role of genetic factors in the development of gastric cancer . Blood tThe study was designed as a case-control study. We set up an active surveillance to identify patients with gastric cancer. Patients' records in the department of pathology in the main private referral facility in Tehran were scrutinized for gastric cancer cases between 1999 and 2003. Patients are referred here from all regions of the country and from different ethnic backgrounds and they are operated upon in the same hospital, so all the operation and pathology reports were available simultaneously. All the pathology reports were prepared and diagnosed by the same pathologists. The cases were selected from patients who were diagnosed with gastric cancer before the age of fifty. The sex-matched controls were randomly selected and enrolled from patients who were diagnosed over the age of fifty. All the patients and their family were interviewed regarding the history of gastric or other types of cancer over three generations, and the blood groups of affected members were ascertained. Family histories of cancer were accepted as valid provided that they were based on valid medical documents. The transfusion records of the operation were also used to identify the patients' blood groups.Statistical analysis was performed using the SPSS Statistical Package (version 10.0). The quantitative variables were expressed as means (minimum-maximum) when appropriate. A chi-square test was performed to ascertain the overall effect of blood group on the development of gastric cancer before the age of 50. All statistical tests were two-sided and differences at the 0.01 level were considered statistically significant.At the beginning of the study, 44 cases  under 50 years old and 44 sex-matched controls (mean age: 67.1, 50–88) were enrolled. Table This case control study demonstrates that hereditary factors, especially familial history of cancer and possession of blood group O, are associated with the development of gastric cancer under the age of fifty. To our knowledge, this may be the first study showing a correlation between blood group O and the development of gastric cancer in a specific category of patients.Risk factors for gastric cancers have been explored in a number of previous studies, including genetic factors such as blood group. Haenszel et al. suggested an association between gastric cancer and blood type A, supporting the view that genetic factors have a role in the development of gastric cancer . Our stuIn our study, approximately 54% of the case group had a familial history of cancer compared to 11% of the control group. This seems compatible with the findings of a population-based case-control study of stomach cancer in Warsaw, Poland. Here, the investigators interviewed 464 cases and 480 controls to evaluate the role of family history and other risk factors. A greater than threefold increase in risk was associated with a history of gastric cancer in a first degree relative (OR = 3.5), but no excess risk was seen for other forms of cancer. The risk associated with familial occurrence was not significantly modified by gender, age or ABO blood type, and did not vary with Lauren histological classification24. Despite the relatively large sample size in the Polish study, younger patients were not evaluated as a separate category. This may explain the difference between their results and ours. Furthermore, they defined "positive family history" as having a first-degree relative with gastric cancer. In contrast, we considered all types of malignancy in first and second-degree relatives; though the familial incidence of other (non-GI) malignancies in our study may reflect their higher frequency in the community rather than any genetic risk factor. The Polish study did not confirm previous results on the correlation between blood group A and gastric cancer. Moreover, another study by Parsonnet et al on 90 cases and 89 controls showed no association between ABO blood group and malignancy . In a muMatching the geographical origins of the cases and controls could have improved the power of our study by excluding ethnic factors from the study population. However, this is very difficult to achieve in such studies because there is a high rate of combinations between races in the country. In addition, the sample size was too small for such effects to be excluded. However, there were no significance differences between the two groups in respect of the origins of the subjects.In conclusion, our results show that familial history of cancer, and hereditary factors including blood group, have a role in the development of gastric cancer in young patients. The role of environmental factors may be more important in older patients and can be considered in future studies.The authors declare that they have no competing interest.MY designed and assisted in the conduction of the study, analyzing of the data and draft the manuscript.NR, FS, RB and YJ assisted in the conducting and designing the study and interview with the study cases as well as drafting manuscript.AM and MA assisted in analyzing of the study and conducting interviews.AA and MH reviewed and approved the pathology reports of the patients and case collecting.RM supervised the study scientifically and executively and assisted in drafting the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Surrogate measures for cardiovascular disease events have the potential to increase greatly the efficiency of clinical trials. A leading candidate for such a surrogate is the progression of intima-media thickness (IMT) of the carotid artery; much experience has been gained with this endpoint in trials of HMG-CoA reductase inhibitors (statins).We examine two separate systems of criteria that have been proposed to define surrogate endpoints, based on clinical and statistical arguments. We use published results and a formal meta-analysis to evaluate whether progression of carotid IMT meets these criteria for HMG-CoA reductase inhibitors (statins).IMT meets clinical-based criteria to serve as a surrogate endpoint for cardiovascular events in statin trials, based on relative efficiency, linkage to endpoints, and congruency of effects. Results from a meta-analysis and post-trial follow-up from a single published study suggest that IMT meets established statistical criteria by accounting for intervention effects in regression models.Carotid IMT progression meets accepted definitions of a surrogate for cardiovascular disease endpoints in statin trials. This does not, however, establish that it may serve universally as a surrogate marker in trials of other agents. Atherosclerosis is a generalized disease that causes lesions in large- and medium-sized elastic and muscular arteries. As lesions progress, arterial walls are remodeled, a process through which the size of the arterial lumen is preserved. Because of this, the disease is clinically asymptomatic during its earlier stages and may go unnoticed for decades as the risk for its clinical manifestation as acute vascular disease grows ,2. EpideThe use of surrogate markers for atherosclerosis extent and progression is widespread. Currently, the most established of these is based on carotid intima-media thickness (IMT) as measured by B-mode ultrasound. It is a natural extension to consider these measures as surrogate markers for cardiovascular disease clinical endpoints ,5. If thBoth clinical and statistical criteria for surrogacy have been proposed.Boissel, et al. lay out criteria that markers must meet to be considered as valid surrogates for clinical endpoints . We grouB1: (Efficiency) The surrogate marker should be relatively easy to evaluate, preferably by non-invasive means, and more readily available than the gold standard. The time course of the surrogate should precede that of the endpoints so that disease and/or disease progression may be established more quickly via the surrogate. Clinical trials based on surrogates should require fewer resources, less participant burden, and a shorter time frame.B2: (Linkage) The quantitative and qualitative relationship between the surrogate marker and the clinical endpoint should be established based on epidemiological and clinical studies. The nature of this relationship may be understood in terms of its pathophysiology or in terms of an expression of joint risk.B3: (Congruency) The surrogate should produce parallel estimates of risk and benefit as endpoints. Individuals with and without vascular disease should exhibit differences in surrogate marker measurements. In intervention studies, anticipated clinical benefits should be deducible from the observed changes in the surrogate marker.Prentice views surrogacy as a statistical property and defines it with mathematical expressions ,8. Four P1: The intervention should affect the distribution of T.P2: The intervention should affect the distribution of S.P3: The distribution of T should be dependent on S.P4: Endpoint T should be conditionally independent of Z given S, i.e. S should fully account for the impact of Z on T.This definition may be specific to a particular setting and cohort; a marker may meet the criteria for surrogacy for one intervention, but fail criteria for others. The criteria for surrogacy are based on explicit models, and may also be dependent on covariates and additional explanatory factors being collected and incorporated into these models.These clinical and statistical definitions require different approaches to establish surrogacy, neither of which is clear-cut. To meet the criteria outlined by Boissel, et al. , experieB-mode ultrasound imaging technology has evolved to the extent that the walls of superficial arteries can be imaged non-invasively, in real-time, and with high resolution. Unlike angiography or 'luminology', ultrasound imaging can visualize the arterial wall at every stage of atherosclerosis, from 'normal' arterial wall to complete arterial occlusion. Arterial wall thickness can therefore be measured as a continuous variable from childhood to old age, in patients and healthy controls . StudiesWe are interested in examining the potential of carotid IMT to serve as a surrogate marker for cardiovascular events, in particular cardiovascular mortality, myocardial infarctions, and clinical stroke. The clinical and statistical arguments for surrogacy are contextual, i.e. are based on specific relationships and mechanisms. It is unreasonable to make open-ended claims that a carotid IMT is a surrogate for these endpoints for all interventions and all cohorts, a point that has not been emphasized sufficiently.Our specific focus is to examine surrogacy in clinical trials of HMG-CoA reductase inhibitors (statins). Empirical evidence is largely drawn from statin clinical trials conducted on cohorts of adults at elevated risk for cardiovascular endpoints. Our choice of statins is based, in part, on the many published trials available for these agents. We acknowledge that it is quite possible that IMT may be a valid surrogate for cardiovascular events with respect to statins (i.e. accounting for the effects of these agents on cardiovascular events), but may not be a valid surrogate for other agents  or endpoints. We used Medline searches to identify seven placebo-controlled clinical trials of statins that report both IMT outcomes and cardiovascular events  and events  represent different measurement scales, thus it is difficult to argue they are influenced by statin therapy to quantitatively similar degrees. We drew evidence that the impacts are qualitatively similar using a meta-analysis of the clinical trials listed in Table The four criteria of Prentice  are as fThere is convincing evidence, some of which is summarized in the meta-analysis described above, that statin therapy reduces the risk of cardiovascular events, to the extent that this is now an indication for their use.As noted above, this association is supported by the results of our meta-analysis Table  and elseThe considerable evidence of this association has been discussed above.We know of no published literature that examines this conditional independence for statin therapies. Such a study is difficult to mount as it requires both sufficient power to demonstrate the relative impact on IMT progression of an intervention and sufficient size and follow-up time after this demonstration to assess the ability of measured IMT progression to account for subsequent risk. The only published account to examine the conditional independence of cardiovascular events given carotid IMT is for colestipol-niacin therapy in the Cholesterol Lowering Atherosclerosis Study (CLAS) clinical trial . The 2-yIn our meta-analysis, when IMT progression is included as a covariate in regression models linking cardiovascular disease events to statin treatment, the relative odds ratio is mediated from 0.48 (as tabulated below) to 0.64 and is no longer statistically significant (p = 0.13). This suggests that changes in IMT may account for some, but not all, of the effect of statins on cardiovascular events (i.e. a PTE of 0.3). Several issues complicate this argument, however. Even if a surrogate successfully meets Prentice's criteria for surrogacy within individual trials, because designs, cohorts, and endpoints vary it is to be expected that a surrogate would only account for some, not all, of treatment effects in regression models across trials. Secondly, like many markers, IMT is subject to measurement error and that is not insubstantial. This measurement error, if uncorrected, may lead to marked underestimates of relationships : measureWe have examined, in a structured and rigorous manner, the evidence that carotid IMT progression may serve as a surrogate for cardiovascular disease endpoints in statin trials. Each of the criteria for surrogacy described by Boissel, et al. appears to be met. The first three of Prentice's criteria are met, and the fourth is met by the one published study for which it can be evaluated . Meta-analyses of statin trials provide support for Boissel's criteria and the first three of Prentice's criteria, and are not inconsistent for Prentice's fourth criterion.It is possible that these arguments may generalize to other agents whose mechanisms are similar to statins, however additional analyses, based on criteria for surrogate outcomes, would be required to make this extension.MAE received an honorarium from Sankyo Pharma, Inc for a meeting during which ideas for this manuscript were developed. He is an occasional consultant to other companies concerning the design of clinical trials involving carotid ultrasonography. DHO serves on data safety and monitoring boards for Pfizer and Astra/Zeneca and serves as consultant to Sankyo Pharma and to Merck. JGT received an honorarium from Sankyo Pharma, Inc for a portion of this work. TO has no competing interests. GE has received an honorarium and consulting fees from AstraZeneca Pharmaceuticals for assistance in the planning and implementation ofa clinical trial involving carotid ultrasonography and statin therapy. He also serves as an occasional consultant to other companies on the design and conduct of trials involving carotid ultrasonography in which statins may be included as background therapies, but are not part of the experimental intervention. HM is an occasional consultant to Sankyo Pharma, including attending the meeting during which ideas for this manuscript were developed. He very occasionally consults with other Pharma companies like MSD, Essex, and Lilly. He is a consultant to Boston Scientific for interventional cardiology and intravascular ultrasound and is also a regular teacher in carotid stenting for the Guidant and Cordis companies.MAE, DHO, and HM conceived and drafted this manuscript. MAE, JGT, and TM organized and conducted its meta-analysis. GE provided oversight to analyses and contributed to interpretation of results. All authors read and approved the final manuscript.


Chorioamnionitis is a common underlying cause of preterm birth (PTB). It is hypothesised that polymorphisms in immunoregulatory genes influence the host response to infection and subsequent preterm birth. The relationship between histologic chorioamnionitis and 22 single nucleotide polymorphisms in 11 immunoregulatory genes was examined in a case-control study.IL1A, IL1B, IL1RN, IL1R1, tumour necrosis factor (TNF), IL4, IL6, IL10, transforming growth factor beta-1 (TGFB1), Fas (TNFRSF6), and mannose-binding lectin (MBL2) were genotyped by polymerase chain reaction and sequence specific primers. Multivariable logistic regression including demographic and genetic variables and Kaplan-Meier survival analyses of genotype frequencies and pregnancy outcome were performed.Placentas of 181 Caucasoid women with spontaneous PTB prior to 35 weeks were examined for histologic chorioamnionitis. Polymorphisms in genes IL10-1082A/-819T/592A (ATA) haplotype [Multivariable Odds ratio (MOR) 1.9, P = 0.05] and MBL2 codon 54Asp allele (MOR 2.0, P = 0.04), were positively associated with chorioamnionitis, while the TNFRSF6-1377A/-670G (AG) haplotype (MOR 0.4, P = 0.03) and homozygosity for TGFB1-800G/509T (GT) haplotype (MOR 0.2, P = 0.04) were negatively associated.Sixty-nine (34%) women had histologic evidence of acute chorioamnionitis. Carriage of the IL10, MBL2, TNFRSF6 and TGFB1 may influence susceptibility to chorioamnionitis.These findings demonstrate that polymorphisms in immunoregulatory genes  Escherichia coli [Acute infection of the amniotic fluid and chorioamnion is a common cause of preterm delivery before 34 weeks gestation. Consistent with previous reports , an Austhia coli . The intMBL2 gene that profoundly influence circulating levels of functional, multimeric MBL. These genetic variants and low MBL levels are associated with risk and severity of infection in a variety of clinical contexts [The innate host defense system is also an important factor in pregnancy and the body's response to infection. Mannose-binding lectin (MBL), a protein of the complement system, opsonises pathogens independently of antibody for phagocytosis and influences inflammatory pathways . There acontexts .Fas, a cell surface receptor of the TNF/nerve growth factor superfamily, mediates apoptosis after binding Fas ligand . ActivatIL1 +3953 and TNF -308 are associated with human chorioamnionitis in in vitro studies [TNF -308 A allele has been reported as a risk factor for clinical chorioamnionitis in pregnant women [The immune system operates as a complex regulatory network of cytokines and other mediators with a degree of redundancy that changes as pregnancy progresses . This ma studies  and carrnt women . HoweverWe hypothesized that polymorphisms in immunoregulatory genes may influence susceptibility to chorioamnionitis and subsequent preterm labour. To examine this hypothesis, we investigated multiple polymorphisms in immunoregulatory genes in a population of Australian Caucasoid women experiencing spontaneous PTB before 35 weeks and histologic chorioamnionitis.This case-control study was approved by the Women's and Children's Hospital Ethics and Research Committee. A total of 368 unrelated women of child-bearing age who had obstetric management at the Women's and Children's Hospital (WCH) and history of PTB before 35 weeks gestation were identified from the hospital data base. All women were approached in the first instant by letter of introduction and invited to participate in the study. For women living in the Adelaide metropolitan area, the study nurse made a home visit to those who volunteered for eligibility assessment. Geographical distance excluded 50, 66 did not respond, 40 were not eligible and 31 declined. Of the women enrolled some had more than one PTB. The PTB fitting the study criteria and closest to the enrolment date became the index birth. Written consent was obtained and a 9 ml peripheral blood sample was collected for isolation of leucocytes and DNA extraction. 181 women with a history of spontaneous preterm labour and subsequent PTB between 20 and 35 weeks in the index pregnancy had histological examination of the placenta performed. Histopathology staff members were unaware of the genetic results.Clinical data recorded included gestational age at delivery . Obstetric factors known to place a woman at risk of preterm labour or preterm prelabour rupture of membranes (PPROM) (defined as prelabour rupture of membranes 3 or more hours before preterm delivery) were also recorded. Placental examination and swabbing was performed by staff histopathologists  and inclDemographic characteristics, a comprehensive medical and obstetric history, age, any smoking, alcohol consumption, substance use and clinical chorioamnionitis (defined as any three of the following – white blood cell >1500 × 10^9/L, three consecutive C reactive protein readings ≥ 15mg/L, pyrexia ≥ 38°C, uterine pain or tenderness) were verified from case records. Clinical chorioamnionitis was not used as a subgroup for polymorphic analysis because this did not include women with sub-clinical infection. Since the risk of chorioamnionitis is not as great in PTB after 35 weeks, women with PTB between 35 and 37 weeks were not enrolled .MBL2) [TNF, IL4, IL6, IL10, TGFB1, TNFRSF6, and MBL2 genes and their relationship with histologic chorioamnionitis were therefore examined in a cohort of women with spontaneous PTB.Genes were chosen if reported to be associated with inflammatory disease and if eMBL2) , thus muTNF (+488,-238,-308) [IL1A (-889), IL1B (+3962, -511), IL4 (-590), IL10 (-1082, -819, -592), TGFB1 (-800, -509) [MBL2 [-550, -221, codon 52 (Arg → Cys), codon 54 (Gly → Asp), codon 57 (Gly → Glu)] [IL6 (-174), IL-1 receptor antagonist IL1RN (+11100), IL-1 type 1 receptor IL1R1 (+970) and TNFRSF6 (-1377, -670) [IL10, TNFRSF6, TNF, TGFB1 and MBL2 genes by PCR haplotyping. The term "haplotype" denotes ordered combination of alleles on a single chromosome, and is used where multiple polymorphisms are genotyped by PCR-haplotyping for a single gene. For PCR haplotyping combinations of forward and reverse allele-specific-primers were used to directly amplify alleles on the same chromosome [DRB1 (major histocompatibility complex, class 11 DR beta 1) and APC (adenomatous polyposis coli) genes were included in every PCR mix to verify successful amplification. All genes except the IL1A had a repeat genotyping rate of less than 1%. The IL1A had a repeat rate of 10%.Peripheral blood samples were blinded and tested at the Australian Red Cross Blood Service, Adelaide (ARCBS). Genomic DNA was extracted from ethylenediaminetetraacetic acid anticoagulated venous blood using standard methods as previously described . SNPs fo38,-308) , IL1A (-0, -509) , MBL2 [- → Glu)] , IL6 . We estimated the study had sufficient power (1-β = 80%, P = 0.05) to detect a minimum of 17% difference in genotype frequency between chorioamnionitis and no chorioamnionitis for frequencies of approximately 10% (ie. 10% versus 27%). For a genotype frequency of 3% the minimum detectable difference was 10% (3% versus 13%). Allele and haplotype frequencies for polymorphisms were determined by direct counting from each chromosome. Frequencies of genetic variants were recorded as allele (proportion of positive chromosomes), genotype , and 'carriage' of an allele or haplotype (either in homozygous or heterozygous state). Alleles were said to be in Hardy-Weinberg equilibrium if the observed genotype frequencies did not differ significantly (P > 0.05) from those expected when analysed by Chi square. Gene variant frequencies were compared using 2 × 2 contingency table analysis and the Chi square test for independence using EpiInfo 6. Mean birth weights were compared by Multivariable analysis was performed by stepwise backwards multiple logistic regression using SYSTAT 9.0 software (Systat version 1999 SPSS Inc). The basic model included genetic variables with P values < 0.15 obtained in univariate analyses. In each multivariable model, only one genotype or haplotype of each gene was selected for analysis. Selection of genotype/haplotype for the model was based on strength of association, the previously reported importance of a gene variant and homozygosity of alleles. At each backward step in the regression analysis P values ≥ 0.07 were rejected. Kaplan-Meier survival analysis (SYSTAT 9.0) was used to identify cumulative probability in maintaining pregnancy by gestation stratified by chorioamnionitis and gene variables. Breslow-Gehan Chi square was used to weight each gestational period by the total number at risk at that time so that earlier gestations receive greater weight than later gestations.Sixty-nine (38%) women had histologic evidence of acute chorioamnionitis, and 112 (62%) had no such evidence. There were no significant differences between those with and without histologic chorioamnionitis for maternal age, race, history of smoking, alcohol consumption, substance use, new partner, cervical cerclage, placenta praevia after 22 weeks with bleeding, history of two or more PTBs before 35 weeks, miscarriage between 12 and 20 weeks or PPROM (Table IL10-1082A/-819T/-592A (ATA) haplotype [MBL2 codon 54Asp  [TNFRSF6-1377A/-670G (AG) haplotype [TGFB1-800G/-509T (GT) homozygosity [All alleles were in Hardy-Weinberg equilibrium. All gene variants were tested for associations between the two groups. In preterm women without histologic chorioamnionitis frequencies for the aplotype , MBL2 co allele) , TNFRSF6aplotype  and TGFBzygosity  were simIL10-1082A/-819T/-592A (ATA) haplotype (present in 49% of women with chorioamnionitis vs 33% without chorioamnionitis, OR 2.0, P = 0.03) and the IL10-819T and -592A alleles (50% versus 33%, OR 2.0, P = 0.03) were positively associated with histologic chorioamnionitis, as was carriage of the MBL2 54Asp  (39% versus 25%, OR 1.9, P = 0.04) (Table TNFRSF6-1377 G/G genotype was higher in women with histologic chorioamnionitis (0.87 versus 0.73, OR 2.4, P = 0.03), the variant TNFRSF6-1377A/-670G (AG) haplotype (OR 0.4, P = 0.03) and homozygosity for the TGFB1 haplotype -800G/-509T (GT) (OR 0.2, P = 0.03) were negatively associated with chorioamnionitis  and IL1R1 +970 T/T (6% versus 14%, OR 0.4, P = 0.08) from IL-10 ATA haplotype [Multivariable Odds Ratio (MOR) 1.9, P = 0.05)] and MBL2 codon 54Asp (MOR 2.0, P = 0.04) were independently associated with histologic chorioamnionitis, while homozygosity for the variant TGFB1-800G/-509T (GT) haplotype (MOR 0.2, P = 0.04), and carriage of TNFRSF6-1377A/-670G (AG) haplotype (MOR 0.4, P = 0.03) remained negatively associated with histologic chorioamnionitis. The remainder of SNPs tested showed no significant differences.For confirmation of independence of association, a multivariable model was used including univariate variables with P values ≤ 0.15. This model included the above genetic variables Table  plus TNFIL10 ATA haplotype   Figure . There wIL10 ATA haplotype, MBL2 codon 54Asp and histologic chorioamnionitis, and negative associations with TNFRSF6 and TGFB1. This is the first study to investigate TNFRSF6 and TGFB1 in combination with such a wide range of cytokine SNPs in histologic acute chorioamnionitis. Also, this is one of the few studies that have used multivariable and survival analyses for investigating adverse pregnancy outcomes such as chorioamnionitis and PTB.These results extend prior reports investigating gene polymorphisms and the pathogenesis of PTB and chorioamnionitis . In thisIL10 and MBL2 may be of biologic importance in the pathogenesis of histologic chorioamnionitis. The function of IL-10 in pregnancy is of particular interest because of its role in the homeostatic control of an inflammatory immune response [IL10 ATA promoter haplotype and alleles comprising the haplotype are associated with low protein levels in Caucasians [IL10 ATA haplotype may influence the timing of PTB. IL-10 antagonises the synthesis and actions of pro-inflammatory cytokines such as IL-1 [IL10 ATA haplotype may also be a reflection of failure of tocolysis in the presence of infection [IL10 ATA haplotype is more common in women with histologic chorioamnionitis suggests that this haplotype may be important in the pathogenesis of histologic chorioamnionitis and subsequent PTB.The differences in genotype distribution for response . Althougucasians . Low levucasians . In a prucasians . Surviva as IL-1 . In chor as IL-1  and feta as IL-1  signal fnfection . Our finMBL2 variant alleles in exon 1 correlate with decreased circulating MBL. The single nucleotide substitution of an adenine for a guanine in codon 54 results in replacement of aspartic acid for a glycine in the MBL [MBL2 peptide trimers resulting in lower stability and serum levels [MBL2 genetic variants are associated with infection in a range of contexts including recurrent infection in children and adults [MBL2 codon 54Asp and histologic chorioamnionitis suggests that this allele may result in low MBL levels, impaired opsonisation and clearance of pathogens, thus facilitating the development of chorioamnionitis.MBL is an important innate defense molecule active against a broad range of bacterial, viral, fungal and protozoan pathogens . The MBL the MBL . Functiom levels . Low serd adults ,14, recud adults , recurred adults  and autod adults . The assTNFRSF6) gene promoter were studied at positions -1377 and -670, and the -1377A/-670G (AG) haplotype was negatively associated with histologic chorioamnionitis. The -1377G/A is situated at the transcription factor SP-1 binding site [TNFRSF6 transcription, Fas expression and thus apoptotic and inflammatory responses. Recent evidence suggests that cells are more sensitive to Fas-mediated apoptosis when levels of Fas expression increase [Fas protein is expressed on the surface of many cell types, such as lymphocytes, epithelial, fibroblasts and certain endothelial cells, and cytokines  and horming site , and theincrease . Apoptotincrease  and thusTNF promoter -308A allele was associated with clinical chorioamnionitis in a small number of women [in vitro functional data of the effects of the TNF SNPs are conflicting and may be context dependent and influenced by strong linkage disequilibrium with other intra-and-extra-genic polymorphisms  declare that they have no competing interests.MA contributed to study design, recruited study subjects, abstracted medical records, entered data, performed preliminary statistical analyses and drafted the manuscript.PH provided design regarding immunoregulatory genes, interpretation of results and preparation of manuscript.CM conceived methodology and supervision of PCR testing, interpretation of test results and preparation of manuscript.SH supervised preparation of DNA and performed all PCR tests.JR provided clinical authorisation and support for the study. Provided input regarding clinical database design, and provided critical analysis for obstetric correctness.HM conceived and coordinated the study, conceived medical record review, reviewed statistical analyses and preparation of manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:Gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation and histologic chorioamnionitis. Frequency distribution of immunoregulatory gene alleles, haplotypes and genotypes in Caucasoid Australian women with preterm birth before 35 weeks gestation stratified by histologic chorioamnionitis.Click here for file