Spirituality has become a subject of interest in health care as it is was recognized to have the potential to prevent, heal or cope with illness. There is less doubt that values and goals are important contributors to life satisfaction, physical and psychological health, and that goals are what gives meaning and purpose to people's lives. However, there is as yet but limited understanding of how patients themselves view the impact of spirituality on their health and well-being, and whether they are convinced that their illness may have "meaning" to them. To raise these questions and to more precisely survey the basic attitudes of patients with severe diseases towards spirituality/religiosity (SpR) and their adjustment to their illness, we developed the SpREUK questionnaire.In order to re-validate our previously described SpREUK instrument, reliability and factor analysis of the new inventory (Version 1.1) were performed according to the standard procedures. The test sample contained 257 German subjects (53.3 ± 13.4 years) with cancer (51%), multiple sclerosis (24%), other chronic diseases (16%) and patients with acute diseases (7%).As some items of the SpREUK construct require a positive attitude towards SpR, these items (item pool 2) were separated from the others (item pool 1). The reliability of the 15-item the construct derived from the item pool 1 respectively the 14-item construct which refers to the item pool 2 both had a good quality . Factor analysis of item pool 1 resulted in a 3-factor solution  which explains 53.8% of variance. Factor analysis of item pool 2 pointed to a 2-factor solution  which explains 58.8% of variance. Generally, women had significantly higher SpREUK scores than male patients. Univariate variance analyses revealed significant associations between the sub-scales and SpR attitude and the educational level.The current re-evaluation of the SpREUK 1.1 questionnaire indicates that it is a reliable, valid measure of distinct topics of SpR that may be especially useful of assessing the role of SpR in health related research. The instrument appears to be a good choice for assessing a patients interest in spiritual concerns which is not biased for or against a particular religious commitment. Moreover it addresses the topic of "positive reinterpretation of disease" which seems to be of outstanding importance for patients with life-changing diseases. Spirituality has become a subject of interest in health care, and an increasing number of studies, commentaries and reviews examine the connection between religiosity/spirituality and health, its potential to prevent, heal or cope with diseases -10. MoreHowever, although religiosity and spirituality were interchangeable words, these constructs may not be identical. It is well established to divide Religiosity into three sub-constructs: Intrinsic, Extrinsic, and Quest Religiosity -20, whilSpiritual Well-Being Scale [Daily Spiritual Experience Scale [Santa Clara Strength of Religious Faith Questionnaire [Functional Assessment of Chronic Illness Therapy – Spiritual Well-Being (FACIT-Sp) scale has a much more open design [The measurability and operability of spirituality and religiosity remains a problem and thus several questionnaires address this topic. Most of them measure beliefs of specific religious groups, and ask about the relationship with God  and their adjustment to their illness, we developed the SpREUK questionnaire ,29,36-39In this article we report the re-validation of the SpREUK 1.1 questionnaire , an instrument designed to examine attitudes of patients with life-threatening and chronic diseases towards spirituality/religiosity.All individuals were informed of the purpose of the study, were assured of confidentiality, and gave informed consent to participate. The patients were recruited consecutively in the cancer service, the multiple sclerosis service, and two internal medical units of the Communal Hospital in Herdecke (Germany). All subjects completed the questionnaire by themselves. Demographic information is provided in Table The sample contained 257 subjects of whom 70% were women. The mean age was 53.3 ± 13.4 years. The majority had a Christian nomination (80%), 17% had no religious orientation, and 3% other nominations. Cancer was diagnosed in 51%, multiple sclerosis in 24%, and other chronic diseases in 16% ; 7% of the individuals were patients with acute diseases . Patients in final stages of their disease were not enrolled.The items of the SpREUK 1.0 were developed with the patients' input  and experts' statements  ,36, rathAccording to a previously conducted reliability and factor analysis ,37 the SIn order to more precisely differentiate the three topics guidance, control and message of disease in scale 2 of the version 1.0, for the current version of the questionnaire, six new items were added . All items were scored on a 5-point scale from disagreement to agreement . The SpREUK scores are referred to a 100% level (4 "applied very much" = 100%).Reliability and factor analysis of the new inventory were performed according to the standard procedures. Next, to combine several items with similar content, we relied on the technique of factor analysis which examines the correlations among a set of variables, and to achieve a set of more general "factors." Factor analyses were repeated rotating different numbers of items in order to arrive at the solution which demonstrated both the best simple structure and the most coherence.Differences in the SpREUK scores were tested using the Kruskal-Wallis-Test for asymptomatic significance. We judged p < 0.05 significant, and 0,05 < p < 0.10 as a trend.To tested the impact of several variables on the SpREUK sub-scales, we performed analysis of univariate variance (ANOVA). As in several cases Levene's test for equality of variances was significant, and we judged p < 0.01 as significant.All statistical analyses were performed with SPSS for Windows 10.0.In order to eliminate items from the item pool that were not contributing to the questionnaire reliability, the reliability of the scale and distinct sub-scales was evaluated with internal consistency coefficients, which reflect the degree to which all items on a particular scale measure a single  concept.Our item pool consisted of the previously established set of items ,36,37 anReliability analysis revealed that 6 items from the new item pool 1 had a poor corrected item-total correlation and thus were eliminated : F1.2 , F1.3 , F2.1 , F2.2 . F2.3 , and F3.1 . One item (F3.6 "The "true being" ("inner core") can not be affected by illness") was omitted because of a weak reliability (0.2997) and – even more important – it points to a distinct "field of meaning" that would need more items in the questionnaire, and thus will be used as marker item until the construct will be revised for this topic.As shown in Table Thus, the internal consistency of the 29-item SpREUK 1.1 construct was sufficiently high. The level of difficulty  is 0.6205 for item pool 1 resp.  0.6014 for item pool 2. With the exception of item F3.7 , all values are in the acceptable range from 0.2 to 0.8.To combine several items with similar content, we relied on the technique of factor analysis which examines the correlations among a set of variables, and to achieve a set of more general "factors." Factor analyses were repeated rotating different numbers of items in order to arrive at the solution which demonstrated both the best simple structure and the most coherence.With a Kaiser-Mayer-Olkin value of 0.850 (item pool 1) resp. 0.939 (item pool 2), which measures the degree of common variance, the 15 resp. 14-item-pool seems to be suitable. Barlett's test for non-sphericity was highly significant .Primary factor analysis of item pool 1 pointed to a 5-factor solution. However, due to a low item number in the tentative subscales 2–5 (with 2 or 3 items each), we favoured the more appropriate 3-factor solution which explains 53.8% of variance  reveal that items F1.8 ("looking for purpose and meaning in life") load good on sub-scale 2 (0.414). Analysis of the side-loadings of item pool 2 revealed that several items load also on the other sub-scale. Moreover, sub-scale 4 showed a strong but negative inter-correlation with sub-scale 5 , the lowest for sub-scale 1 ("Search for meaningful support"). Means and standard deviations for study variables are provided in Table Women had significantly higher SpREUK scores than male patients. With respect to age, the lowest SpREUK scores were found in the group of < 30 years of age. With increasing age, the trust in a higher supporting presence  and the beneficial effects of resp. support through SpR increased.With respect to the marriage status, widowed patients obviously has to rely on external guidance  but not the patients living with a partner not married with. Widowed and divorced patients find support in external relations through their SpR engagement , while – in contrast to married patients which may find hold in their partnership – especially divorced patients are in search for meaningful support .Search for meaningful support and positive interpretation of diseases were depending on the educational level, as patients with lower educational level had significantly lower scores than those with a higher level. A higher educational level was associated with higher scores in the sub-scales 4 and 5 which deals with the beneficial effects of SpR.Illness itself (but not the duration of disease) has a significant impact on the SpREUK scores, as MS patients had the lowest scores in all 5 sub-scales. The SpREUK scores of cancer patients revealed slight differences when compared to patients with other chronic diseases.With the exception of sub-scale 2, patients without confessional affiliations had the lowest scores for all sub-scales, indicating that the "message of disease" was not depending on a denomination. Surprisingly, the few patients with other than a Christian orientation had the highest scores for sub-scales 1, 3, 4, 5.(R+S+); 35% as religious, but not spiritual (R+S-); 23% as neither religious nor spiritual (R-S-); 10% claimed that they were spiritual, but not religious (R-S+). Thus, the numbers of patients with denominational affiliation and self-reported spiritual/religious attitudes is somewhat similar. A spiritual attitude (R+S+ and R-S+) was associated with "search for meaningful support" and "positive interpretation of disease", while a religious attitude (R+S+ and R+S-) was associated with the highest scores for the "trust in external guidance" sub-scale 3.Since nominational affiliation is not necessarily identical with religiosity or spirituality, we asked whether the patients would describe themselves as religious or spiritual ,34,35. TThe living area and the duration of diseases had no significant impact on the SpREUK scores.As shown in Table In detail Table , prayingNext we tested the impact of several variables on the SpREUK sub-scales, such as sex and marital status, educational level and confession, age and SpR attitude, and disease and duration of disease. Using the method of univariate analyses of variance we identified several sources of variability Table :SpR attitude is an important covariate for the "Search for meaningful support", "Positive interpretation of disease", "Trust in external guidance", and both "Support through SpR" sub-scales.• The educational level is an important covariate for "Search for meaningful support", "Trust in external guidance" and to a minor content for "Support in relations with the External life through SpR" – but not for the "Positive interpretation of disease".• The Age is an important covariate only for "Trust in external guidance".• Confession is an important covariate for "Trust in external guidance".• Disease itself has an impact on the "Search for meaningful support• Data from the current analysis demonstrate the reliability and validity of the SpREUK construct. Moreover, the sub-scales 1 and A (= 4) and B (= 5) of the preliminary version 1.0 were confirmed in the new version 1.1. In order to more precisely differentiate the three topics guidance, control and message of disease from the SpREUK version 1.0, six new items were added. Due to this fact, some items from the original item pool decreased the reliability of the construct and thus, two items from the sub-scale 1 had to be deleted  of disease" has a good correlation with an existentialistic practice, which seems to be of outstanding importance for patients with life-changing diseases. It may be desirable to use such a measure that allows to assess attitudes which are independent of any religion or specific belief.A third strength is that the validation was performed in a sample with at least two different types of life-changing diseases  and a healthy control group.Beyond conceptual boundaries, our instrument differentiates the self-addressed "religious" and "spiritual" attitudes of the patients with life-threatening diseases and heeds their search for support and meaning, and integrates the topic of "meaning in illness". We cannot exclude the possibility that these topics are not relevant for healthy individuals.In future studies we have to correlate our scales with other relevant instruments which measure aspects of SpR. Nevertheless, evaluation of the SpREUK questionnaire indicates that it is a reliable, valid measure of distinct topics of SpR that may be especially useful of assessing the role of non-religious spirituality in health related research. The focus of a larger study is to enrol patients from the highly secular Eastern Europe, and to run longitudinal studies with cancer, multiple sclerosis patients, but also cardiac failure and spinal cord damage.The SpREUK with its additional SpREUK-P manual to measure a patient's engagement in distinct forms of SpR practice is currently available in English and German language.AB conceived the study, designed and developed the questionnaire, performed statistical analysis and drafted the manuscript. TO participated to conceive and design the study, performed additional statistical analysis and helped to draft the manuscript. PFM conceived the study and participated in the design and development of the questionnaire. All authors read and approved the final manuscript.


The principles of Virchov's triad appear to be operational in atherothrombosis or arterial thrombosis: local flow changes and particularly vacular wall damage are the main pathophysiological elements. Furthermore, alterations in arterial blood composition are also involved although the specific role and importance of blood coagulation is an ongoing matter of debate. In this review we provide support for the hypothesis that activated blood coagulation is an essential determinant of the risk of atherothrombotic complications. We distinguish two phases in atherosclerosis: In the first phase, atherosclerosis develops under influence of "classical" risk factors, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. Despite the presence of tissue factor – factor VII complex it is unlikely that all fibrin in the atherosclerotic plaque is the direct result from local clotting activity. The dominant effect of coagulation in this phase is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin.The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation as indicated by an elevated level of plasma C-reactive protein, the result of increased production influenced by interleukin-6. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin and endothelial cell protein C receptor (EPCR) expression. In this phase, the inflammatory drive leads to recurrent induction of tissue factor and assembly of catalytic complexes on aggregated cells and on microparticles, maintaining a certain level of thrombin production and fibrin formation. In advanced atherosclerosis systemic and vascular wall driven coagulation becomes more important and elevated levels of D-dimer fragments should be interpreted as markers of this hypercoagulability. The blood coagulation system comprises three basic elements: platelet adhesion, activation and aggregation, fibrin formation, and fibrinolysis. These elements interact with each other and with the blood vessel wall and under physiological conditions blood flow to tissues is unimpaired by clotting . Under pvenous thrombosis of the lower limbs, stasis, local inflammation on activated vascular endothelial cells induced by adhering leukocytes and platelets and in some cases direct vascular damage, promotes local thrombus formation. In a first episode of venous thrombosis the pre-existing composition of the blood is particularly important where congenital and acquired hypercoagulable factors such as factor V Leiden mutation and oral contraceptives, respectively, act in concert to accelerate clotting . Un. Un30]. The involvement of coagulation in the pathological substrate of atherosclerosis is beyond dispute. For many years pathologists have noted the abundant presence of fibrin in advanced atherosclerosis and this finding has fueled part of the debate on the relevance of fibrin or fibrinogen for vessel wall lesions. Rokitansky and later Duguid proposed the encrustation theory as concept for the role of fibrin in atherosclerosis (reviewed in ). In thiearly event in atherosclerosis, i.e. a small amount of fibrinogen in a thickened intima was demonstrated in a 4 year old boy [-/- background did not have fewer arterial lesions ranging from early lesions to complex fibrous plaques, suggesting that fibrinogen is not an essential molecule for atherosclerosis [-/- x apo(a) crossbred animals [Autopsy data have indicated that fibrinogen accumulation in the vessel wall may be an  old boy . The depclerosis . However animals .The specific effect of fibrin and its split products in the vessel wall has also been studied. In general it appears that with increasing complexity of lesions there is an increase in the presence of intimal fibrinogen/fibrin and threads of fibrin, as well as an accumulation of various split products that may be involved in atherogenesis. The effect of fibrin and its split products on smooth muscle cells may be such that fibrin stimulates proliferation, while split products inhibit this process. Fibrin cleavage products may be detrimental for endothelial cell function, increasing permeability and promoting endothelial cell migration ,41,42. D-/-) results in markedly discrepant effects on atherosclerosis. While Plg-/- mice with an apoE-/- background showed an accelerated development and progression of intimal lesions, Plg-/- mice were protected against atherosclerosis in association with transplantation . The orAccumulating fibrin that polymerizes in the vessel wall triggers fibrinolysis. Fibrinolytic enzymes tissue plasminogen activating factor and urokinase plasminogen activating factor  are present in intima and are secreted by endothelial cells and likely play an important role in vascular remodeling -44. Howe-/- mice give important clues regarding the range of mechanisms that are influenced. Effective fibrinolysis may be important in limiting fibrin accumulation and atherosclerosis in the initial phases. However, upon stronger inflammatory stimulation the effect on cell trafficking into the vessel wall becomes more dominant and the outcome may reverse such that impaired fibrinolysis may limit atherosclerosis. The latter would imply that high levels of PAI-1 may even be protective against atherosclerosis under certain conditions. Consequently, high concentrations of D-dimers, reflecting active fibrinolysis, may indeed be regarded as a sign of progressive atherosclerosis under inflammatory conditions.The net effect on fibrin cleavage and progression of atherosclerosis cannot be estimated. The above mentioned experiments with PlgAll of these issues may have therapeutic consequences since several drugs that are routinely used in patients with atherosclerosis including statins, angiotensin converting enzyme inhibitors as well as angiotensin receptor blockers appear to influence the balance of coagulation and fibrinolysis, which may influence atherosclerosis on the long term by altering vascular properties .As mentioned above, a large number of clinical studies in different groups of patients with atherosclerotic disease have generally shown that increased levels of D-dimer fragments in plasma are associated with an increased risk of severe atherosclerosis and an increased risk of vascular complications. In population based studies the contributable risk of an increased D-dimer level is quite small but statistically significant. In specific cohorts of patients the risk association is more outspoken, but of course here selection bias may produce slightly stronger associations than may be found in "real life".Before addressing specific study findings a few general observations deserve attention.First, strong associations between age and sex on the one hand, and D-dimer levels on the other hand, are noted ,11,42. DSecond, D-dimer levels are oftentimes associated with markers of inflammation, i.e. CRP and Il-6 ,25,42,46Before addressing the mechanisms we will consider D-dimer as an independent entity, i.e. a marker of disease severity. The most striking associations with clinical disease come from patients with peripheral artery disease (PAD), a reflection of systemic and advanced atherosclerosis in the majority of individuals. The total risk of clinical complications or mortality reaches figures of up to 25% annually in patients with PAD 48). In patients with PAD, elevated D-dimer levels are independent predictors of complications and are associated with severity of atherosclerosis -14,16,25. In patiElevated levels of D-dimers are usually considered as a marker of increased clotting activity. This assumption is one of the key elements of the controversy regarding cause and consequence of hypercoagulability. Indeed, Herren et al observed increased levels of D-dimer in patients with PAD, correlating with severity of disease. They also noted an association between hypercoagulability (higher F1+2 and TAT) and occurrence of myocardial ischemia during exercise testing , suggestsystemic clotting activity then a specific anticoagulant intervention may theoretically be the preferred intervention. Clinical studies with anticoagulants in patients randomized or stratified on the basis of D-dimer levels have, however, not been carried out yet.In spite of the substantial observational data, application of D-dimer assays or other risk factor measurements such as for CRP have not gained acceptance in individual patients with PAD or other cardiovascular disease yet. Thus, secondary prevention of complications is not guided by any laboratory assay, but limited to general recommendations such as the advice to stop smoking and the prescription of a platelet inhibiting drug . Three rseverity of atherosclerosis such an intervention may be inappropriate and potentially harmful because of the avoidable risk of bleeding and calcification of the arterial vessel wall upon long-term administration (at least with vitamin K antagonists). Alternatively, if D-dimer levels merely reflect inflammation, than therapy should preferably consist of anti-inflammatory agents including higher doses of aspirin, statins or ACE inhibitors. Thus, the interpretation of elevated D-dimer levels is quite important in order to guide decisions about individual therapy.If however, D-dimers are a reflection of Atherosclerosis is a chronic inflammatory disease ,28. ThisStudies from the sepsis field including models of endotoxemia and sepsis in humans and primates, respectively, have shown that inflammatory stimulation leads to activation of blood coagulation ,53. TissIn this concept, there is an increased generation of thrombin and fibrin in the blood circulation due to increased presence of inflammatory cytokines and proteins, but this does not necessarily lead to increased free thrombin in plasma. One should realize that coagulation enzymes that are generated associate with any available "scavenger", which can be an inhibitor such as antithrombin, but could also be a protease activated receptor (PAR) on platelets or endothelial cells -56. ThusThe exact contribution of subendothelial fibrin formation and cleavage to D-dimer levels in blood cannot be estimated. While locally deposited tissue factor acts as a trigger of thrombin generation, it has not been shown that this is a source of ongoing subendothelial coagulation activity. The recent discovery of factor VII in plaque contents and the in vitro evidence for production of this Gla-protein by smooth muscle cells might form a basis for local thrombin production, but there is no indication yet that this might be quantitatively important as compared to hepatic production of factor VII .systemic hypercoagulability, a conclusion based on the above arguments and the experimental evidence indicating the intimate relationship between inflammation and coagulation [The point to make is that high D-dimer levels in blood from patients with atherosclerosis should be primarily viewed as an indication of gulation .Early studies from Rosendaal et al, suggested that thrombophilic traits including the prothrombin 20210 gene variant would be a risk factor for myocardial infarction in specific individuals such as heavy smoking young women . These dIn people of advanced age this situation may change considerably and the weight of risk factors may change over time. A study that specifically addressed this point is the Bruneck community study . In thisIn advanced atherosclerosis the influence of coagulation may indeed be more prominent than in early stages, but it should be realized that acquired rather than genetically determined forces are involved. In this regard, the similarities between arterial thrombosis and the risk of recurrent venous thrombosis was used by Reitsma to make the point that inflammation is a key player under such conditions, reducing the influence that genetic thrombophilic background might inflict. On the other hand the same argument could be used to illustrate that indeed inflammation plays a more prominent role in advanced atherosclerosis where it more strongly drives the risk of thrombosis.Let us consider this situation from the scope of venous thrombosis; this is not far edged because a recent study suggested similarities in risk factors between patients with previous venous thrombosis and atherosclerosis . Recent The influence of blood coagulation on atherosclerosis follows a two stage model in which variants may occur under exceptional conditions. In general in the first phase, roughly covering the first four decades of life, atherosclerosis develops under influence of "classical" risk factors, including hypercholesterolemia and smoking, i.e. both genetic and acquired forces. While fibrinogen/fibrin molecules participate in early plaque lesions, increased activity of systemic coagulation is of no major influence on the risk of arterial thrombosis, except in rare cases where a number of specific procoagulant forces collide. The dominant effect of coagulation is anticoagulant, i.e. thrombin enhances protein C activation through its binding to endothelial thrombomodulin. Defects in the protein C mechanism may indeed precipitate arterial thrombosis, but only under highly thrombogenic conditions. Fibrinolysis limits fibrin accumulation in the intima and herewith progression of plaque lesions. At this stage elevated PAI-1 levels may diminish fibrinolysis and may stimulate plaque progression, which may explain that in a large Japanese study the PAI 4G/5G polymorphisms appeared to be a risk factor for myocardial infarction in women .The second phase is characterized by advancing atherosclerosis, with greater impact of inflammation and increased infiltration of fibrin in the arterial vessel wall, enforcing pro-inflammatory effects. The extensive interactions between inflammation and coagulation enzymes and inhibitors (in much greater detail than discussed here) amplify the chain of events that determine the risk of atherothrombosis. Inflammation overwhelms protective anticoagulant forces, which in itself may have become less efficient due to down regulation of thrombomodulin (TM) and endothelial cell protein C receptor (EPCR) expression. In this phase, evidence of activated coagulation is measurable in peripheral blood reflecting both the extent of atherosclerotic burden and the systemic clotting tendency, which poses a direct risk of thrombotic complications. This point of view deviates from Tracy's viewpoint and provides a more constructive model for integrating coagulation in arterial disease.We would also propose that D-dimer and other, novel assays such as for endogenous thrombin generation   or for a


Recently, intracardiac echocardiography emerged as a useful tool in the electrophysiology laboratories for guiding transseptal left heart catheterizations, for avoiding thromboembolic and mechanical complications and assessing the ablation lesions characteristics. Although the value of ICE is well known, it is not a universal tool for achieving uncomplicated access to the left atrium. We present a case in which ICE led to interruption of a transseptal procedure because several risk factors for mechanical complications were revealed.A case of a patient with paroxysmal atrial fibrillation and atrial flutter, and distorted intracardiac anatomy is presented. Intracardiac echocardiography showed a small oval fossa abouting to an enlarged aorta anteriorly. A very small distance from the interatrial septum to the left atrial free wall was seen. The latter two conditions were predisposing to a complicated transseptal puncture. According to fluoroscopy the transseptal needle had a correct position, but the intracardiac echo image showed that it was actually pointing towards the aortic root and most importantly, that it was virtually impossible to stabilize it in the fossa itself. Based on intracardiac echo findings a decision was made to limit the procedure only to ablation of the cavotricuspid isthmus and not to proceed further so as to avoid complications.This case report illustrates the usefulness of the intracardiac echocardiography in preventing serious or even fatal complications in transseptal procedures when the cardiac anatomy is unusual or distorted. It also helps to understand the possible mechanisms of mechanical complications in cases where fluoroscopic images are apparently normal. Since the advent of ICE in the electrophysiology practice it proved its value in guiding transseptal procedures with providing an extra safety margin for the patients. The possibility to visualize the oval fossa, the LA free wall and the aortic root helps in preventing mechanical complications. ICE can visualize also intracardiac thrombus and spontaneous echocontrast, which is helpful in avoiding thromboembolic complications. Although the value of ICE is well known, it is rather hard to admit that it is a universal tool for achieving uncomplicated access to the left atrium. The aim of this case presentation is to show that ICE can lead to interruption of a transseptal procedure due to the presence of risk factors for mechanical complications when the fluoroscopic image is seemingly satisfying.A seventy-year-old male patient with paroxysmal atrial fibrillation and atrial flutter, and concomitant arterial hypertension was referred to our institution for LA circumferential ablation. The preprocedural TEE described an aneurysm of the IAS See . During In the last years ICE emerged as a useful adjunctive tool in the field of interventional electrophysiology. It serves not only scientific purposes but practical issues as well. Its value for achieving successful and uncomplicated transseptal access to the LA cavity is well known -4. HowevIn this case ICE showed that the aneurysm of the IAS observed during the TEE was actually the angulated continuity between the enlarged aortic root and the IAS. The echocardiographic orientations of ICE are sometimes clearly off axis in comparison to standard transesophageal echocardiographic views. Nevertheless, to our opinion ICE is superior in providing more detailed picture of the neighboring cardiac structures. Although useful in guiding transseptal catheterizations, TEE does not always provide complete avoidance of complications even in patients with normal hearts ,6. FurthEP procedures are relatively safe procedures and have low complication rate. One of the most frequent complications is related to cardiac wall perforation with consequent pericardial effusion and tamponade. ICE, especially the one with phased-array transducer (deeper penetration) allows continuous monitoring of the pericardial space during EP procedures. This permits prompt detection of a pericardial effusion and an immediate guidance of a therapeutic puncture. Phased-array transducers are equipped with Doppler capabilities allowing assessment of the pulmonary venous flow pattern after pulmonary vein ablation on top of diameter measurements to exclude pulmonary vein stenosis, which is the most important complication of this procedure. Monitoring microbubble formation during RF energy application allows prevention of pulmonary vein stenosis . ICE alsWe strongly believe that this case is a good illustration of the usefulness of the ICE in the electrophysiology field and further enhances its value as a tool for avoiding complications when the intracardiac anatomy is unusual or distorted. As the number of transseptal procedures in the electrophysiology laboratories all over the world is steeply growing ICE definitely has the potential to become a routine at least in those institutions with large volume of left atrial procedures.EP – electrophysiologicIAS – interatrial septumICE – intracardiac echocardiographyLA – left atrium; left atrialTEE – transesophageal echocardiographyPreprocedural transesophageal echocardiography. The irregular oval-shaped structure at the center of the screen is the aortic root. At a certain moment one can see at its upper part the ostium and the most proximal part of the right coronary artery. Below is situated the left atrium and to the left – the right atrium. The oval fossa is in between.Click here for fileIntracardiac echocardiography, showing distorted intracardiac anatomy. The oval shape in the center of the screen is the non-coronary sinus of Valsalva. Below is the right atrium at the bottom of which the transseptal needle is clearly visible. The prominent muscular structure in the left-hand part of the image is the terminal crest. The membrane of the oval fossa, adjacent to the right-hand part of the non-coronary aortic sinus shows bidirectional flapping motion. During the atrial contraction the cavity of the left atrium virtually disappears.Click here for fileIntracardiac echocardiography showing sliding of the needle towards the aorta. The transseptal needle is already in the oval fossa with its tip pointing to the aorta. This is especially clearly visible after a premature beat.Click here for file


Background to the debate: The US and Canadian task forces on preventive health recently declared that there is not enough evidence to recommend for or against routine universal screening of women for domestic violence. Yet some experts argue that routine enquiry is justified. Experts from both sides of the Atlantic debate whether screening can be justified based on the available clinical evidence Domestic violence is a misunderstood topic. The context of a trusted health professional talking to a woman is one that provides an important opportunity for providing information to counter misconceptions.asking all women about domestic violence and not in terms of screening women for domestic violence. It is not appropriate or helpful to regard enquiry about being abused as a form of screening. Domestic violence is not a disease present in the body of the person who experiences it—rather it is a health-related risk factor.I deliberately talk about this in terms of As such, knowledge of abuse puts health professionals in a position to respond better to the needs of women affected by it. Professionals can respond by providing information on specialist services—usually provided outside the health service—that women may access if they wish. By giving information to affected women, health professionals can also help to reduce women's sense of isolation and stigmatisation. Asking about experience of domestic violence can be seen as a routine part of history taking, just as health professionals regularly and repeatedly ask patients about their smoking behaviour, alcohol use, weight, and exercise.The prevalence of domestic violence among women is such that, even if it is not a personal issue for the woman concerned, it most likely will be for one or more of her relatives, friends, and neighbours . Since mMost women experiencing domestic violence report that the specialised services that exist to respond to their needs were difficult to find out about . The proStudies have examined women's views on being asked about domestic violence. These studies have shown that once they have experienced being asked, they are usually in favour of being asked. This is true both for those who have experienced or are experiencing abuse, and those who have not . It is oAsking about abuse should be done in a flexible fashion—the particular questions used should respond to the circumstances of the consultation. For example, it is appropriate to ask women about domestic violence as part of a health check in a Well Woman Clinic, but it would be completely inappropriate in a consultation where another adult or a child was present. By being flexible, health professionals can integrate their questioning within a variety of different encounters. Integrating questions about abuse into routine encounters provides for the maintenance of confidentiality and safety. In order to do this, health professionals require training on raising the issue and knowledge about local advice and support services.Committees on both sides of the Atlantic have rejected the notion of screening women for domestic violence, arguing that there is insufficient evidence of the effectiveness of interventions ,5. Part In one example of an excluded study, researchers used a randomised design to evaluate an advocacy service for women experiencing domestic violence ,8. WomenSystematic reviews have also excluded, or devalued, evidence from qualitative studies. For example, a study of 200 women who had used domestic violence outreach services found that about half were living in situations of domestic violence when they first contacted the service. All of these women reported that the outreach services had helped them to leave the abusive relationship—a valued outcome for them .Given the health impacts on women who experience domestic violence (not to mention their children) and the prevalence of the problem, routinely asking women about abuse should be seen as an important form of primary and secondary prevention for a wide range of health problems.Screening tools for domestic violence are abundant, and many are effective at identifying women experiencing abuse ,10. HoweGiven the morbidity and mortality associated with domestic violence, it is tempting to suggest that universal screening for abuse should be integrated into routine clinical care, such that all women, regardless of their reason for presenting to a clinical setting, should be “asked the question.” Some argue that this approach is justified by the need to increase awareness of domestic violence as a significant problem with serious health and social consequences, and to make abused women aware that they are not alone in their experience. These are important considerations.Certainly all women who disclose that they have been exposed to violence should be provided with options regarding seeking help . Good diwithout obvious signs and symptoms of domestic violence—such as a woman who comes to the clinic for assessment of an upper respiratory tract infection? Should such women be prompted to disclose whether they are being abused? The woman who is not being abused will answer to that effect, and the appointment can carry on. But for the woman who is experiencing violence, who has not volunteered this information, several factors must be considered. An important issue is whether she is ready—both psychologically and in terms of taking specific actions—to confront the issue. A number of excellent qualitative studies have examined the process that women undertake in acknowledging that they are “victims” of “abuse” and embarking on the often long and difficult journey to avoid, reduce, and ultimately stop the violence in their lives [However, what about women presenting ir lives ,17. GiveAny potential benefits of screening must then be weighed against its potential harms, including labelling women, prompting potentially premature disclosure, and triggering possible reprisal violence from the abuser if he discovers she has sought help. The last of these might be particularly exacerbated for the woman with the respiratory tract infection who was unprepared to disclose and did not take necessary precautions. Other potential harms include exposure to the ramifications of laws on mandatory child protection reporting, whereby health providers must report such disclosures to child protection authorities. This can lead to an investigation that potentially increases a woman's risk of exposure to violence, and in some cases of having her children placed in foster care. Research has shown that many of these potential harms are of concern to women when mandatory universal screening and/or reporting protocols are in place . FinallyGiven the lack of clear data on the benefits of screening and of the interventions to which women are referred, and the lack of data on potential harms, we and others have concluded the following ,19,20. UI agree entirely with Nadine Wathen and Harriet MacMillan that practice should be based on evidence. There are further areas of agreement. We agree that there is a lack of knowledge on effective interventions for abusers and on harm occurring as a result of enquiry, and that targeted case finding is important.all women about domestic violence. Underlying this difference is the issue about how much evidence we need, and of what type. My position is that the evidence that already exists is sufficient to justify the promotion of routine enquiry, aiming to ask all women about their experience of abuse. There is evidence of actual benefits to women—and their children—from interventions provided by specialised services for domestic violence and from brief discussions with health professionals [The key difference that exists between my viewpoint and theirs is the conclusion about whether health professionals should aim to ask ssionals .Aiming to ask all women has several advantages over targeted case finding . It contThe twin issues of women's safety and harm minimisation are extremely important, for both routine enquiry and targeted case finding. These issues are important reasons why training and protocols for enquiry are necessary. Standard principles of confidentiality should be reinforced in training and protocols, which need to be tailored to relevant legal requirements, such as when child protection issues are involved. Training and protocols also need to emphasise that the role of routine enquiry is to facilitate, and not force, disclosure. It must remain the woman's choice as to if, when, and to whom, she discloses.all women or only in situations where asking about it is part of a specific diagnostic assessment. As with screening for a disease, universal screening for domestic violence should not be implemented unless we are sure that interventions are available to help those identified via screening and that screening plus appropriate treatment will do more good than harm.We agree with Ann Taket that domestic violence is not a disease, and that the paradigm of “screening for disease” is problematic in this context. At issue, however, is the question of whether domestic violence should be “talked about” with Professor Taket outlines the importance of integrating discussions about abuse in consultations to raise community awareness. Unfortunately, there is no evidence that this type of consciousness-raising occurs, or if it does, what benefit it might have. Given the lack of effectiveness of educational campaigns in general, it is difficult to be optimistic about this approach.We disagree with her conclusion that existing systematic reviews have “excluded studies done outside the health service setting….” Our review included interventions such as the post-shelter advocacy counselling approach to which Professor Taket refers . This inFinally, we concur that qualitative studies are invaluable in understanding domestic violence. Such research has provided insight into the complex process that women undertake to address the violence in their lives. Until there is evidence that universal screening actually helps with this process, the focus should be on developing evidence-based approaches to assist women when they do disclose abuse and on training health professionals to respond appropriately to such disclosures.


The cellular signaling pathway (network) is one of the main topics of organismic investigations. The intracellular interactions between genes in a signaling pathway are considered as the foundation of functional genomics. Thus, what genes and how much they influence each other through transcriptional binding or physical interactions are essential problems. Under the synchronous measures of gene expression via a microarray chip, an amount of dynamic information is embedded and remains to be discovered. Using a systematically dynamic modeling approach, we explore the causal relationship among genes in cellular signaling pathways from the system biology approach.In this study, a second-order dynamic model is developed to describe the regulatory mechanism of a target gene from the upstream causality point of view. From the expression profile and dynamic model of a target gene, we can estimate its upstream regulatory function. According to this upstream regulatory function, we would deduce the upstream regulatory genes with their regulatory abilities and activation delays, and then link up a regulatory pathway. Iteratively, these regulatory genes are considered as target genes to trace back their upstream regulatory genes. Then we could construct the regulatory pathway (or network) to the genome wide. In short, we can infer the genetic regulatory pathways from gene-expression profiles quantitatively, which can confirm some doubted paths or seek some unknown paths in a regulatory pathway (network). Finally, the proposed approach is validated by randomly reshuffling the time order of microarray data.Arabidopsis thaliana and metabolic shift pathway from fermentation to respiration in yeast Saccharomyces cerevisiae, are reconstructed using microarray data to evaluate the performance of our proposed method. In the circadian regulatory pathway, we identified mainly the interactions between the biological clock and the photoperiodic genes consistent with the known regulatory mechanisms. We also discovered the now less-known regulations between crytochrome and phytochrome. In the metabolic shift pathway, the casual relationship of enzymatic genes could be detected properly.We focus our algorithm on the inference of regulatory abilities of the identified causal genes, and how much delay before they regulate the downstream genes. With this information, a regulatory pathway would be built up using microarray data. In the present study, two signaling pathways, i.e. circadian regulatory pathway in  Biological phenomena at different organismic levels have revealed some sophisticated systematic architectures of cellular and physiological activities implicitly. These architectures were built upon the biochemical processes before the emergence of proteome and transcriptome -3. Underin situ synthesized oligonucletide  and Cry2 [X10] are commonly regulated by Lhy [X3] (LATE ELONGATED HYPOCOTYL) in reciprocal ways with significant values (0.7569 in Eq.(6) and -1.8773, Eq.(10) of Table Lhy on crytochrome genes. In addition, from Eq.(10) in Table Ccal [X4] (CIRCADIAN CLOCK ASSOCIATED 1) has the greatest positive regulation (2.3465) on Cry2, meaning that Cry2 is jointly regulated by Lhy and Ccal. Because the binding sites of Lhy and Ccal found in the promoter regions of Cry2  and PhyA [X7] in Eq.(6) and Eq.(7) of Table Cry2 [X10] is also positively regulated by PhyA [X7] with 0.5-hr activation delay similar to that in Cry1 (Eq.(6) in Table PhyA is considered as a post-transcriptional regulator of phosphorylation to crytochrome within 1.0-hr after transcription. On the other hand, PhyB [X11] down-regulates Cry2 with a significant effect (-0.7141) while Cry2 [X10] up-regulates PhyB (0.0511) weakly by feedback (see Eqs.(10), (11) in Table Cry2 and PhyB in nuclear speckles that are formed in a light-dependent fashion are also confirmed by Mas et al. [Cry1 and Cry2 are both negatively co-regulated by PhyD [X8] and PhyE [X12] significantly (see Eqs.(6), (10) in Table PhyA has apparently different behavior from PhyB, PhyD, and PhyE in activating crytochrome. This might suggest the mechanism that PhyA mediates the blue light by up-regulating Cry1 and Cry2, whilst PhyB, PhyD, and PhyE would mediate the red light by inhibiting blue photoreceptors  and Ccal [X4], well-known biological clock genes in the circadian system [Gi [X15] (GIGANTEA) in Figure Gi sequence lacks any motifs suggesting that it is a transcription factor of phytochromes  (EARLY FLOWERING 3) opposite to Gi on phytochrome, especially for PhyA, PhyB and PhyE (Eqs.(7), (11) and (12) in Table Lhy or Ccal, Elf3 might play the same role as Lhy and Ccal. Just as expected, Elf3 contains glutamine-rich motif suggesting that it is a transcription factor  (0.1223) (see Eq.(9) in Table Toc1 [X13] is transcriptionally regulated by Lhy [X3] (0.7009) and Ccal [X4] (-1.4704) whilst Pif3 [X16] (-0.1698) is presumably considered as the bridge between Toc1 and phytochrome (Eq.(13), Table Lhy and Ccal point of view, they are both positively affected simultaneously by Pif3 implying the regulation on the transcriptional level  Figure  is mediaal level . In addiChs [X5] (CHALCONE SYNTHASE), Pap1 [X1], and Co [X2] (CONSTANS) in Figure Chs is known as correlated with UV-B protection. It seems that Ccal and Lhy have greater effect  on Chs than Pap1 (-0.0455) as a transcription factor (Eq.(5) in Table Chs is regulated by Pap1 in a small scale with amplifying effect on the cis-regulatory level. Co is recognized as a pivotal gene of photoperiodic regulation of flowering. Indeed, strong regulations from Ccal and Lhy are identified to show that Co is regulated with a large-scale attenuation effect on the cis-regulatory level (Eq.(2) in Table We also infer some downstream pathways of Chs X , represented by black lines with more linking to upstream genes, the photo-transduction pathways are stabilized to provide oscillation. On the other hand, more blue lines of inhibitive interactions are revealed in the biological-clock regulatory pathways relevant to Co, Pap1, and Chs  underlying the anti-phase functional regulation between these output pathways and the oscillator. In addition, the essential signal transduction factors of Fkf1, Gi, Elf3, and Pif3  make some critical links between the functional blocks mentioned above in the circadian system encoding an enzyme, which catalyzes PEP (Phosphoenolpyruvate) to pyruvate, is negatively regulated (-5.8763) by Pck1 [Y26] (Eq.(4) in Table Pck1 could be intepreted here as an indirect upstream transcription factor or regulatory gene for Pyk1 due to its function of decarboxylation and phosphorylation of oxalacetat in the presence of a nucleoside triphosphate and a divalent metal ion to yield PEP. Another Gcrl [Y15] gene is also identified as the strongest positive regulation (5.9829) to Pyk1  in Table Gcrl of Pdc1 [Y6] (PYRUVATE DECARBOXYLASE ISOZYME 1) plays a more essential role  in Table Rap1 [Y17] (0.1164), and Pyk1 [Y4] is an upstream regulatory factor coding an enzyme with the most positive effect (3.1295) on Pdc1 according to the production of acetaldehyde from pyruvate. In the last kernel of the fermentation, Adh1 [Y7] and Adh2 [Y3] are involved in the ethanol metabolism of carbohydrate storage. Adh2 is implicated to up-regulate Adh1  in Table Adh1  in Table Gcr2 [Y16] and Sfp1 [Y30] with consistently dominant negative influences on Adh2 and Adh1 respectively would be at the transcriptional level presumably (see Eqs.(3), (7) in Table In the fermentation direction, by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)by Pck1 Y6 (Eq.(4)Eno2 [Y2] (ENOLASE ISOZYME 2) is regulated by Pck1 [Y26] (-0.7195) in the same way as Pyk1 while the main transcription factor is Stp2 [Y31] with significantly positive regulation  in Table Eno2, Gpm1 [Y8] (PHOSPHOGLYCERATE MUTASE l), Tpi1 [Y10] (TRIOSE-P ISOMERASE 1), Fba1 [Y11] (ALDOLASE 1), and Pgi1 [Y9] (PHOSPHOGLUCOSE ISOMERASE 1) indicates the construction of a trunk of the glyconeogenesis (see Eqs.(8), (9), (10), and (11) in Table Rap1 [Y17] and Gcrl [Y15] are the common regulators of Gpm1, Pgi1, and Tpi1. This means that Rap1 and Gcrl might be the most important regulators in the glyconeogenesis pathway by the transcriptional binding. Finally, Pgm2 [Y5] (PHOSPHOGLUCOMUTASE 2) co-regulated by Glk1 [Y27] (GLUCOKINASE 1), Hxk1 [Y28], and Hxk2 [Y29]  significantly confers another pathway leading to the synthesis of UDP-GLU from Glucose-6-P (see Eq.(5) in Table In the sub-pathway of glyconeogenesis, Adh1 and Adh2 are apparent when compared with the obscure relationships in glyconeogenesis . Interestingly, three transcription factors Gcr1, Gcr2 and Rap1  appear to have very significant effects on the metabolic shift pathway. Finally, in order to validate the proposed method, an independent validation is also given by randomly reshuffling the time order of microarray experiment but with same choices of target gene and regulatory gene, as shown in Figure In the overview of the metabolic shift pathway in Figure Microarray expression analysis by the dynamic system approach offers an opportunity to generate functional regulation interpretation on the genome-wide scale. The crucial ontology behind using dynamic system techniques is that the causality between gene expression profiles could be identified according to the differential equation underlying a dynamic system. Therefore, because the microarray data were harvested with time progression, the simultaneously varied gene expressions implicated in a genetic regulatory system would be detected to infer the regulatory pathways in spite of the versatile interactions such as transcriptional control, protein phosphorylation, or specific enzyme regulation.The clustering method answers the problem of what is the functional catalogue of a specific gene by the identification of resembling patterns of gene expressions. Similarly, the co-regulations of upstream genes in our method also imply their concurrent functions. In contrast to the clustering algorithm, the causality of time-course data has been smoothly drawn by our dynamic method. The Bayesian networks were used merely for forward probabilistic estimation with time transition lacking in the feedback linkages. This unidirectional problem would not happen in our algorithm. Owing to the quantitative regulatory abilities of our model, we have a greater diversity of regulatory influence than the Boolean networks, which are deterministic with merely two states.In our dynamic system approach, we not only can link qualitatively the upstream genes to the downstream ones iteratively, but also indicate quantitatively their regulatory relationships, including the regulatory abilities and the activation delays. In terms of the regulatory abilities, the comparison between the upstream regulatory genes of a target gene can inspire us to ask which one is significant biologically and whether it is a positive or negative influence on the investigated gene. Moreover, the speculation of activation delays benefits the empirical reference by providing us when the upstream regulatory genes might interact with their target genes. Since any gene can be considered as a target gene to trace back its upstream regulators, these regulators are then considered as target genes to trace back their upstream regulators. Iteratively, the genetic regulatory pathway (or network) can be constructed to the genome-wide. According to the qualitative and quantitative features imbedded, two regulatory pathway examples are characterized as in Figure In the two pathways under investigation, we have a more detailed understanding about the regulatory interactions among phytochrome, crytochrome and biological clock in the circadian regulatory system. On the other hand, the sophisticated knowledge of the metabolic pathway after the diauxic shift can be unfolded properly in our analysis. Furthermore, the independent validation of our approach is also given by randomly reshuffling the time order of microarray experiment. We found that the proposed pathways in Figures There are some shortcomings in our study. First, although the time-course microarray data are available, its lower samplings will distort the real changes of gene expressions, especially for quick dynamic evolution. A more sampling experiment with respect to the intrinsic turnover rate is expected to have more precise analysis. Secondly, a regulatory gene with larger activation delay would not be recognized because the less activation delay criterion is used, but this might be overcome by properly relaxing the criterion. Thirdly, activation profiles under the proteome should be highly correlated with the transcriptional profiles to elevate the interpretation of our system model. In general, the synchronous time-course microarray assay is more suitable to underlie the transcriptional binding among causal genes, but an inference of physical interactions in the post-transcriptional level also has sufficient feasibility in our study.In the near future, the most pressing task is to investigate our presumed paths in the laboratory. As the pathway construction algorithms are further developed, we expect this system approach to have immense impact in elucidating the underlying molecular mechanisms of pathways in a variety of organisms, especially after the maturation of the protein chips. Ultimately, we envision that biologists will perform routine pathway inference to seek some novel regulations and to identify the evolutionarily conserved links.Gi(t) in equation (1.2), we substitute equation (1.2) into equation (1.1) to obtain the following dynamic equation for the expression profile of the i-th gene,After the decomposition of ai, bi, αn, and βn should be estimated by the time profile of microarray data of the i-th gene, i.e. these parameters should be specified so that the simulating output Xi(t) of the dynamic model in equation (3.1) should meet the empirical expression profile of the i-th gene. The least-squares estimation method is employed to solve this parameter estimation problem.In the above dynamic equation, parameters t = t1,…, tm and is then arranged in a vector differential form. Consequently, the vector differential form underlined in this equation is applied to m time points in order.To make the dynamic model effective, the dynamic equation in (3.1) should meet the expression profile at all time points , , and m denotes the number of time points.where In the next step, formula (3.2) can be translated into a differential matrix equation as follows,Yi = AiΦi + Ei     (3.3), Φi = [ai bi α0 β0 … αN βN]T, and  are in vector forms, while  is a matrix.where i, the least-squares method below is used to derive the optimal parameters estimation of ,To estimate the relevant unknown parameters in ΦEi as the noise of the gene-expression profile or of the microarray chips. So the consideration of modeling error makes equation (3.3) approach more the reality. By the way, in order to get accurate data of  and  from the expression profile of the target gene, the cubic spline should be employed to interpolate the time profile of the target gene. Furthermore, the choice of N is based on the tradeoff between the accuracy of approximation in (1.2) and the complexity of parameter estimation in (3.4). In this study N = 6 is chosen because these harmonics are enough to approximate regulation functions.Actually, the modeling error could be concluded into i in equation (2.4) is given as follows:Maximum likelihood method for Ωm data points is then described byThe log-likelihood function for given σ2 is , by which equation (2.5) is obtained.The necessary condition for the maximum likelihood estimation of variance Substituting equation (2.5) into equation (4.2) yields,i by minimizing the value of σ2 in equation(2.5). Then, the maximum likelihood estimate in equation (2.6) is obtained by  in equation(2.5).meaning that we can find the maximum likelihood estimation of ΩW.C. Chang and Chang-Wei Li carried out the computational studies and analysis. B.S. Chen gave the topic and suggestions. All authors read and approved the final manuscript.The raw data has been transform to .mat file, which is one of the Matlab files. This file has been divided into three parts: Name, Time, and Profile. see  Name denClick here for fileThe 'Shuffled_data' is the Shuffled raw data. see Click here for file


As anorexia and hypermetabolism are common in cirrhosis, leptin levels may be increased in this disease. In this study, we investigated the relation between the severity of disease and serum leptin levels in post-hepatitis cirrhosis and the role of body composition, gender and viral aetiology of cirrhosis in this association.Thirty-five cases with post-hepatitis cirrhosis and 15 healthy controls were enrolled in this study. Body composition including body mass index, body fat percentage and body fat mass were determined. Serum leptin levels were assayed.Leptin levels were significantly higher among cirrhotic patients independent of sex compared to controls (p = 0.001). Female patients in both groups have had higher leptin levels than males .Cirrhotic patients in each of A, B and C subgroups according to the Child- Pugh classification revealed significantly different levels compared to controls . Male cirrhotics in Child-Pugh Class B and C subgroups had significantly higher leptin levels compared to male controls . On the other hand, female patients only in Child Pugh class C subgroup have had higher levels of serum leptin compared to controls (p = 0.022).Child-Pugh classification has been found to be the sole discriminator in determination of leptin levels in cirrhotics by linear regression (beta: 0.435 p = 0.015).Serum leptin levels increase in advanced liver disease independently of gender, body composition in posthepatitic cirrhosis. The increase is more abundant among patients that belong to C subgroup according to the Child- Pugh classification. Leptin, a 16-kilodalton protein, is involved in the regulation of food intake and body composition  and has In normal humans, circulating level of leptin is higher in women than in men ,5. BesidMalnutrition is a common feature of cirrhotic patients . A negatIn this study, we investigated the relation between the severity of disease and serum leptin levels in post-hepatitis cirrhosis and the role of body composition, gender and viral aetiology of cirrhosis in this association.Thirty-five cases with post-hepatitis cirrhosis  which were diagnosed on the basis of the clinical, laboratory, radiological, and/or histopathological findings, and 15 healthy controls  were enrolled in this study. Cirrhotic cases were assigned into 3 groups on the basis of the Child-Pugh classification  as folloControl group consisted of healthy individuals with normal medical history, physical examination and blood biochemistry. None of them have had a restriction of diet for loosing weight during the last three months. Subjects who receive any medication have not been included into control group. The local human institutional review committee approved the study and written consents were received from all participants.th percentile  compared to controls  .Gender based serum leptin levels of controls and cirrhotic cases that were grouped according to Child Pugh Classification were as shown in Figure When age, gender, BFM, hepatitis B and C virus as etiologic factors of cirrhosis and child A, B and C as Child-Pugh classification were tested as independent variables for determination of serum leptin levels as dependent variable by linear logistic regression analysis in cirrhotic group, analysis result showed that Child-Pugh classification was the sole discriminator in determination of serum leptin levels in cirrhotic cases  Table .Leptin regulates body weight by suppressing appetite and increasing energy expenditure ,3,4. AnoLeptin levels are higher in woman than in men ,6. McCulSince BMI and BFM values do not differ according to the sex and the presence or absence of cirrhosis, increased serum leptin levels could not be simply dedicated to BFM or malnutrition status in cirrhosis. In addition, linear regression test in the present study has shown that disease severity, which was determined by Child-Pugh classification, was the sole significant determinant of serum leptin levels in cirrhosis. In previous studies, association between the severity of cirrhosis and serum leptin levels is controversial -13. HenrIn an animal study, it has been shown that chronic ethanol consumption leads to increased serum concentrations of tumor necrosis factor and related cytokines such as leptin by inducing over production of these factors in the liver and peripheral adipose tissues . Leptin Serum leptin levels increase in advanced liver disease independently of gender, body composition and viral etiologic factor in post-hepatitis cirrhosis. The increase is more abundant among patients that belong to C subgroup according to the Child- Pugh classification.BMI, body mass index; BFP, body fat percentage; BFM, body fat massThe authors declare that they have no competing interests.Bolukbas FF conceived of the study, and participated in its design and coordination. Bolukbas FF, Bolukbas C, Erdogan M and Zeyrek F collected the samples and carried out the laboratory analysis. Bolukbas C conceived of the study and participated in the sequence alignment and drafted the manuscript. Horoz M participated in the design of the study, participated in the sequence alignment and drafted the manuscript. Gumus M collected the clinical data and performed the statistical analysis. Yayla A drafted the manuscript and revised it critically for important intellectual content. Ovunc O participated in study design and coordination and revised the manuscript critically for important intellectual content.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


When publishing large-scale microarray datasets, it is of great value to create supplemental websites where either the full data, or selected subsets corresponding to figures within the paper, can be browsed. We set out to create a CGI application containing many of the features of some of the existing standalone software for the visualization of clustered microarray data.We present GeneXplorer, a web application for interactive microarray data visualization and analysis in a web environment. GeneXplorer allows users to browse a microarray dataset in an intuitive fashion. It provides simple access to microarray data over the Internet and uses only HTML and JavaScript to display graphic and annotation information. It provides radar and zoom views of the data, allows display of the nearest neighbors to a gene expression vector based on their Pearson correlations and provides the ability to search gene annotation fields..The software is released under the permissive MIT Open Source license, and the complete documentation and the entire source code are freely available for download from CPAN  Microarray experiments produce vast amounts of data. The resulting datasets are highly complex and contain large matrices of expression measurements as well as sequence and experiment annotations that provide biological context to the data. To organize these different types of data in a way that allows intuitive exploration of the data, and provides the ability to gain important insights into relationships within a given dataset requires sophisticated visualization tools. Such visualization tools are of benefit not only to researchers analyzing and presenting or publishing their own data, but also to Model Organism Databases (MODs) for compiling and displaying microarray data for a given model organism.There are several excellent free tools available that allow an individual user to analyze their own data. These tools are either accessible on the web, or can be downloaded and used on a desktop machine. Examples include the EPCLUST , GEPAS ,3 and FGThe application was written using object oriented Perl following the Model-View-Controller (MVC) design paradigm . GeneXplThe Microarray::CdtDataset has two essential functions: during dataset creation (see below) it decomposes the data file into its constituent data parts and creates the files needed during data viewing (see below). During data viewing it provides the API for the viewer, and allows searching and retrieval of the data. Under the current model the dataset object itself is immutable. Microarray::CdtDataset was implemented as a client of the Microarray::DataMatrix module, which provides an API for accessing matrices of expression data. In the design of the classes certain compromises had to be made to accommodate the stateless client server environment in which the program operates. Specifically, to allow rapid responses, pre-generated images and correlation data are cached in a compact format on the web-server.There are two stages required to publish a microarray dataset on the web using GeneXplorer. The first stage (executed only once per dataset) involves creation of all the necessary files for GeneXplorer to use. The second stage uses these files to produce the display using the GeneXplorer web front-end.Dataset creation requires a file in the Clustered Data Table (CDT) format: a simple tab delimited text file format  that would allow us to do the updates via web services.The GeneXplorer package is provided as a typical Perl distribution on the Comprehensive Perl Archive Network (CPAN), and adheres to the usual installation mantra of perl modules. After unpacking the software, a user with administrative privileges merely needs to type:perl Makefile.PLmakemake testmake installThis will install the libraries and the executable files that are needed for dataset creation by GeneXplorer into the regular system locations, unless otherwise specified during the first step above. The example in Figure In addition to its use within SMD, GeneXplorer has been used by many publications to provide access to microarray datasets through their web supplements, that can be accessed through SMD's publication page , and wasWe are planning to further develop GeneXplorer to enable it to handle other data formats. Specifically, we would like it to be able to accept data files in MAGE-ML format , which iWe have developed a web-application, GeneXplorer, which allows the visualization of microarray datasets over the Internet using only a web browser. This application has been extremely useful in our experience, where it serves both SMD users during analysis of their data and the public while browsing published datasets.GeneXplorer is available at  under thSMD: Stanford Microarray Database.CAR designed and wrote the initial version of the GeneXplorer. This was extensively re-factored and modularized by JCM (library modules for dataset) and JD (for explorer). DB was involved in the guidance of the early stages of this project. GS wrote the correlations software and the DataMatrix classes, and guided the development of this project. All authors read and approved the final version of the manuscript.


Silene which have an X-Y sex-determining system . We used the X-linked copies to build a genetic map of the X chromosomes, with a marker in the pseudoautosomal region (PAR) to orient the map. The map covers a large part of the X chromosomes—at least 50 centimorgans. Except for a recent rearrangement in S. dioica, the gene order is the same in the X chromosomes of all three species. Silent site divergence between the DNA sequences of the X and Y copies of the different genes increases with the genes' distances from the PAR, suggesting progressive restriction of recombination between the X and Y chromosomes. This was confirmed by phylogenetic analyses of the four genes, which also revealed that the least-diverged X-Y pair could have ceased recombining independently in the dioecious species after their split. Analysis of amino acid replacements vs. synonymous changes showed that, with one possible exception, the Y-linked copies appear to be functional in all three species, but there are nevertheless some signs of degenerative processes affecting the genes that have been Y-linked for the longest times. Although the X-Y system evolved quite recently in Silene (less than 10 million years ago) compared to mammals (about 320 million years ago), our results suggest that similar processes have been at work in the evolution of sex chromosomes in plants and mammals, and shed some light on the molecular mechanisms suppressing recombination between X and Y chromosomes.To help understand the evolution of suppressed recombination between sex chromosomes, and its consequences for evolution of the sequences of Y-linked genes, we have studied four X-Y gene pairs, including one gene not previously characterized, in plants in a group of closely related dioecious species of  Similar processes have been at work in the evolution of sex chromosomes in plants and mammals. A recently evolved plant X-Y system is helping to shed light on these processes Drosophila sex chromosomes and some plants [Silene [Drosophila miranda [Drosophila are excellent for studying the rate and causes of degeneration, but cannot shed light on question (i).Newly evolved sex chromosome systems, such as those in plants  and fishe plants , but note plants ? Second,e plants , and emp [Silene ,7 and fr miranda . Recent Silene. This genus is a model for the study of plant sex chromosome evolution, since the sex chromosomes evolved recently [Studies of the evolutionary divergence of gene pairs on mammalian X and Y chromosomes suggest that recombination between the X and nonrecombining parts of the Y was successively suppressed. In many X-Y systems, including that in mammals, there is a “pseudoautosomal” region (PAR) where the X and Y recombine, and it has been found that DNA sequence divergence between homologous X- and Y-linked genes increases with distance from this region. This pattern has been termed “evolutionary strata” ,10. Partrecently ,13.Silene species  includes S. latifolia, S. dioica, and S. diclinis, which have an X-Y sex-determination system with a male-determining Y [Silene species are hermaphroditic or gynodioecious . Dioecy and sex chromosomes thus probably evolved within this genus [Silene species have n = 12 chromosomes [One group of closely related dioecious mining Y ,14, whilis genus . All dipomosomes , so theromosomes .S. latifolia have recently been identified and sequenced  ceased recombining long before the three dioecious species split, whereas the X and Y copies of SlX1-SlY1 (termed locus 1) continued to recombine until recently. We discuss the implications of these results for the mechanism of recombination arrest between the sex chromosomes.Several sex-linked genes from equenced , allowinS. latifolia cDNA. The SlX3 open reading frame of 575 amino acids encodes a protein sequence similar to calcium-dependent protein kinases (CDPKs) from tobacco, rice, and Arabidopsis thaliana . CDPKs are associated with various kinds of stress responses [S. latifolia [Locus 3 was identified from esponses . Thus, latifolia ,19.S. latifolia and S. dioica [latifolia-dioica, latifolia-diclinis, and dioica-diclinis , but for gene 1 no such sites were found. If, on the other hand, X1 and Y1 diverged after the species split, some sites should differ between the species, but not between X and Y of the same species. This is found for mammalian and bird sex chromosomes, and phylogenetic analysis suggests that some X and Y  genes ceased recombining independently in different taxa [Silene species are very closely related [X1 sequences. However, some Y variants are exclusive to each species; we found five nucleotide variants fixed only in the S. latifolia Y (plus nine indel variants), and ten fixed only in S. dioica Y (plus one indel). Since only 11 S. dioica Y sequences were analyzed, the number of fixed Y variants is probably overestimated, however . Furthermore, in a tree estimated excluding these sites with fixed differences in the Y-linked sequences (as is appropriate for such closely related species), the Y sequences are nested within those of the X of each species  between the X and Y sequences of S. latifolia and S. diclinis (dSX-Y) correlates with the gene's distance from the PAR in the X chromosome genetic map . X-Y synonymous divergence also differs significantly between genes 1 and DD44 (p = 0.01). These results suggest progressive suppression of the recombination between X- and Y-linked alleles of different genes. In S. dioica, the same correlation exists, using the S. latifolia or S. diclinis gene order; thus, the rearrangement probably arose recently in S. dioica, consistent with its absence in the other dioecious species. A recent rearrangement, such as an inversion, after the DD44-X and -Y sequences had diverged for some time, would not affect this gene's X-Y divergence relative to that of gene 1. In mouse species, where rearrangements have occurred, evolutionary strata corresponding to those on other mammalian X chromosomes are still plainly discernible [Synonymous divergence etic map B. X-Y sycernible .(dN) was less than dS for divergence between X and Y sequences  tests for these two genes, the dN/dS difference between Y and X is highly significant . Our observation of similar dS values contrasts with previous analyses [S. diclinis Y3 gene also seems to evolve faster than the other Y3 genes  and moving toward the current PAR. This pattern resembles the “evolutionary strata” for mammalian X-Y gene pairs based on Ks values, a measure of divergence per site similar to dS [dSX-Y values among our four gene pairs is 26% for locus 3 in S. diclinis. This overlaps the values for the mammalian stratum 4 and 3 genes ; these strata are inferred to have ceased recombining between the X and Y 30–50 million years ago (MYA) for stratum 4, and 80–130 MYA for stratum 3, whereas strata 1 and 2 diverged 130–320 MYA [The correlation of ar to dS ,10. Howe–320 MYA ,11.S. latifolia, S. dioica, and S. diclinis X-Y sequence divergence data show that X-Y differentiation was already advanced in the common ancestor of these species, except for locus 1. The maximum synonymous X-Y divergence observed for our genes is approximately 25%, including SlAp3, which probably transposed from an autosome onto the Y soon after the sex chromosomes evolved [MROS3-X/Y, whose Y-linked copy is degenerated [Silene species, the Silene sex chromosomes must have evolved much more recently than mammalian sex chromosomes.The  evolved ; all theenerated . Unless Silene. For the nuclear genes Chs and Adh in the family Brassicaceae, estimated rates are, respectively, 1.4 × 10–8 to 2.2 × 10–8 substitutions per synonymous site per year [Ipomoea [S. latifolia group of species. However, substitution rates for some plant Adh genes are almost ten times slower, particularly for plants with long generation times [DD44 differentiated before the S. dioica–S. latifolia–S. diclinis speciation, whereas gene 1 may have ceased recombining after this, perhaps independently in S. latifolia and S. dioica; no analysis can be done in S. diclinis without diversity data for this species, but suppression of X-Y recombination within this species after its split from the other dioecious species is also possible  suggest that the S. latifolia Y may carry genes suppressing recombination, and should help test whether mechanisms other than inversions contributed to reduction of the PAR.The mechanism suppressing X-Y recombination is unknown. Recombination could be reduced either by inversions (or other major recombination rate changes), and/or by modifiers reducing local crossover rates. The “strata” of different divergence in mammalian sex chromosomes may have resulted from a series of Y inversions disrupting X-Y recombination . Inversinversion . Thus, gn Silene ,33, requSilene. Recombination suppression may be selectively favored to preserve advantageous Y-linked combinations of alleles at different loci, such as genes that are advantageous in males but not in females [The mechanism of recombination reduction between X and Y chromosomes is important for understanding the diversity in loci that recently ceased recombining, such as gene 1 in  females , althoug females ,22.Silene sex chromosomes. Degeneration is likely, since genotypes with a Y but no X chromosome are inviable [MROS3-Y in S. latifolia [Silene Y is uncertain, because most currently known sex-linked genes in these plants were ascertained from a cDNA-based search for Y-linked genes. Bacterial artificial chromosome clone sequencing may provide unbiased comparisons of homologous X- and Y-linked regions, and this has been started in papaya [Silene can also be inferred when dN values in the Y are elevated compared with X lineages. This is seen for the two “old” Silene Y-linked genes, locus 3 and locus 4 . Thus the higher dN in the Y-linked alleles is not due to a higher mutation rate (higher dS) in the Y than the X. Moreover, the Y-linked copy of locus 3 fails to amplify in RT-PCR experiments, and may be degenerated.Our analyses suggest that both reduction of recombination and Y degeneration may be in progress for inviable ,35, but atifolia . The extn papaya . Some de locus 4 . DiffereDD44, and no diversity data have yet been obtained for gene 3. The result of this test was nonsignificant; there is thus no evidence that Y1 and Y4 evolution is driven by selection. There is, however, very low polymorphism in the Y copies, so the test has low power [These observations, plus those for gene 1 , suggestow power .Genetic degeneration is supported by low levels of polymorphism of Y- compared with X-linked genes, taking into account the lower Y effective population size ,22. ThisSilene Y-linked genes? Our analyses suggest that degeneration of the genes studied here is partial, at most, consistent with a recent origin of the Silene sex chromosomes. However, there has probably been enough time for degeneration, since this occurred rapidly for genes on the neo-Y chromosomes of D. miranda [Silene Y. Silene sex chromosomes are more advanced in sex chromosome evolution than in some other plants. The papaya sex-determining region is just a small nonrecombining part of one chromosome, yet there is evidence for considerable differentiation, including addition of repeat sequences and some evidence for gene loss [D. miranda neo-Y chromosome [Buchnera [D. miranda neo-Y [Why is degeneration so slight for our  miranda , which aene loss . More liene loss ) may thuromosome , the birromosome , and in Buchnera . In all da neo-Y ), suggesS. latifolia plants were from Edinburgh  and from Fontainebleau forest (France). S. dioica plants were collected in Corrèze (France). S. dioica plants from the Sherringham population , used for isolation of the ScOpa09 marker, were kindly provided by D.L. Mulcahy . S. noctiflora and S. vulgaris were obtained from the seed collection of the Lyon Botanical Garden . Seeds of S. diclinis were obtained from the seed collection of the Institute of Biophysics in Brno (Czech Republic). Interspecific hybrid Silene diclinis × latifolia plants were generated by pollination of a S. diclinis female with pollen of an MAV line male (S. latifolia) kindly provided by S. Matsunaga . The S. latifolia U9 line, which was used for pollination of the interspecific hybrid, was kindly provided by S. Grant .Genomic DNA was extracted from leaves as described . For RT-S. latifolia by the approach that yielded loci 1  and 4 (lX4/SlY4 ). From alX4/SlY4 , and the walking , using t walking , SoutherS. dioica, S. diclinis, and S. noctiflora or S. vulgaris using primers designed from S. latifolia sequences , were as follows. One incubation at 94 °C for 5 min; 35 cycles of: denaturation at 94 °C for 30 s, annealing at a temperature that depended on the primers for 30 s, and elongation at 72 °C for a time depending on the length of the amplicon  being the probable ortholog of SlY/X1. In S. latifolia, some of the copies have been shown not to be sex-linked . Southern blots were not done in S. dioica or S. diclinis, but RT-PCR reactions in all the dioecious species always amplified a single sequence in the females plus another very similar sequence in males, clearly representing the expected X and Y copies. Thus, for the analyses presented later, this gene can be treated as a single-copy gene, as was also the case in previous mapping work [All genes, except gene 1, are single-copy see . In S. lrn blots , and theing work .S. dioica, S. diclinis, and S. vulgaris were amplified, either by RT-PCR (X copies) or from genomic DNA (Y copies). PCR products were cloned into pGEM-T Easy vector , and multiple clones were sequenced for each gene. Sequencing reactions were carried out with ABI Big Dye Terminator V1.1 DNA sequencing kit, on an Applied Biosystems 3100 sequencer .For locus 3, the 3′ two-thirds of the coding sequences of S. dioica or S. latifolia. We have now confirmed sex linkage of all four loci by segregation analysis in all three dioecious species  were designed from this sequence. In plants grown from seeds of this family, presence or absence of the expected amplified fragment accords exactly with results using the original pseudoautosomal RAPD marker primer OPA09. With our primers, S. dioica plants from the Corrèze population were genotyped by digesting PCR products with the restriction enzyme TaqI; the recombination frequency between the marker locus and sex was approximately 2.5%, confirming the pseudoautosomal location. In S. latifolia, genotyping was done using the same primers and an AluI site polymorphism. Genotype data were analyzed by both three-point and multipoint mapping, using JoinMap version 1.4 [To orient the X genetic map, we used a pseudoautosomal marker. For this, we cloned and sequenced a RAPD fragment incompletely linked to the X chromosome of the pollen donor of the eveloped . The seqsion 1.4 . Thus thhttp://pbil.univ-lyon1.fr/) [The primer sequences were removed before sequence analyses. For each gene, the nucleotide sequences were aligned using the corresponding amino acid sequences as a guide, using ClustalW with the Seaview interface (on1.fr/) . Alignmehttp://pbil.univ-lyon1.fr/) [http://pbil.univ-lyon1.fr/) [http://taxonomy.zoology.gla.ac.uk/rod/treeview.html) [Phylogenetic trees were estimated including all sites except those with gaps by neighbor joining (NJ), maximum parsimony, and maximum likelihood (ML), using Phylo_Win (on1.fr/) . For NJ on1.fr/)  and Treeew.html) .dS and dN site divergences were estimated using PAML 3.13 (http://abacus.gene.ucl.ac.uk/software/paml.html) [http://pbil.univ-lyon1.fr/) [dS and dN are similar under various substitution models   and JaDion1.fr/) . Estimatang 1994 , Yang anang 1994 , and Neiang 1994 , implemeang 1994  using Jaang 1994 .dS or dN of X and Y sequences were compared using HyPhy 0.99  expresses dS values for Y lineages as multiples of values for X lineages: Rsyn = dSY/dSX. In model 2 , Rsyn was constrained to be equal to 1 (dSY = dSX). We compared the ML values by a LR test with model 2 as the null hypothesis. We used the same approach to compare dN values (with dN/dS replacing Rsyn). To compare dN/dS using LR tests, we again defined two models. Model 1 assumed two global variables (dN/dS)X and (dN/dS)Y so that the nonsynonymous rates of branches of the X lineage were expressed in terms of the synonymous rate (dN/dS)X, and similarly using (dN/dS)Y for Y branches (“shared dN/dS”), while model 2  assumed (dN/dS)X = (dN/dS)Y.Values for  species  to polarS. diclinis Y3 evolves faster than other Y3 sequences, we assumed a common Rsyn for S. dioica and S. latifolia, as in model 1 above, but added a further parameter, the dSY/dSX ratio for S. diclinis (model 1*). We compared models 1 and 1* using a LR test as above; we tested dN and dN/dS differences similarly.To test whether DD44, and no diversity data are yet available for gene 3.McDonald-Kreitman tests were done using DNAsp software, version 3.95 . The divS. latifolia by contingency tests, using DNAsp. To infer fixed differences rigorously, we used diversity data within S. latifolia for genes 1, 4, and DD44. For gene 3 no diversity data are yet available; however, because this gene pair has high X-Y divergence, raw divergence values should suffice, so for this gene we estimated numbers of differences from single X and Y sequences.To test for differences in divergence between the X and Y sequences of different genes, we compared numbers of fixed X-Y differences in S. latifolia and S. dioica Y and X sequences, plus one sequence from each of two outgroup species, S. vulgaris and S. conica; this enables us to identify whether the changes were in the X or Y lineages, using parsimony. With global gap-removal, the program unambiguously distinguishes fixed differences, including insertions and deletions, from polymorphisms within species. The outgroup sequences are shorter than the other sequences, so some fixed differences in the S. dioica Y could not be analyzed.To test whether X and Y sequences of gene 1 continued recombining and started to diverge after the dioecious species split, a C program was written to find fixed differences in a set of multiple http://www.lirmm.fr/guindon/phyml.html) [As this dataset includes the first approximately 2,000 sites of gene 1, including coding and intron sequences , a more ml.html) , excludiTable S1(36 KB DOC).Click here for additional data file.http://www.ncbi.nlm.nih.gov/) accession number for AtMSI4 is AF028711; the UniProt/TrEMBL (http://www.ebi.ac.uk/trembl/) accession number the ScOpa09 marker is Q9FW98.The GenBank (


Natural killer (NK) cells represent a first line of defence against a developing cancer; however, their exact role in colorectal cancer remains undetermined. The aim of the present study was to evaluate the expression of CD16 and CD57 [immunohistochemical markers of natural NK cells] in colorectal adenocarcinoma.Presence of NK cells was investigated in 82 colorectal adenocarcinomas. Immunohistochemical analysis was performed, using 2 monoclonal antibodies . The number of immunopositive cells (%) was evaluated by image analysis. The cases were characterized according to: patient gender and age, tumor location, size, grade, bowel wall invasion, lymph node metastases and Dukes' stage.NK cells were detected in 79/82 cases at the primary tumor site, 27/33 metastatic lymph nodes and 3/4 hepatic metastases; they were detected in levels similar to those reported in the literature, but their presence was not correlated to the clinical or pathological characteristics of the series, except for a negative association with the patients' age (p = 0.031).Our data do not support an association of NK cell tissue presence with clinical or pathological variables of colorectal adenocarcinoma, except for a negative association with the patients' age; this might possibly be attributed to decreased adhesion molecule expression in older ages. Colorectal adenocarcinoma is a neoplasm in which prognosis is mainly determined by the histological stage. However, the prognosis of certain patient groups, especially those of intermediate stages, remains vague . The maiNatural killer (NK) cells play a pivotal role in innate immunity and immunological surveillance. Their cytotoxic effects may be initiated without prior immunization and thus NK cells have been considered as a first line of defence of the host against a developing cancer ,3. In spThe aim of this study was to evaluate tissue presence of NK cells in a series of colorectal adenocarcinomas and to attempt to correlate their presence with clinical and pathological variables and prognostic markers of colorectal cancer.From June 1997 to May 2000, 82 patients from our Department underwent colectomy, due to a diagnosis of colorectal adenocarcinoma of conventional histologic type. NK cell presence was examined via standard 3-step immunohistochemical analysis (ABComplex), which was performed on formalin-fixed, paraffin-embedded tissue sections. Briefly, two appropriate monoclonal antibodies were used  at dilutions of 1/200 with overnight incubation. Antigen retrieval was necessary and was performed by usual microwave treatment. In the examined samples, tissue identification of NK cells was based on strong CD16 immunostaining, which was frequently accompanied by CD57 immunoreactivity. Tissue sections from hypertrophic tonsils were used as positive markers. Diaminobenzidine tetrahydrochloride 0.06% in phosphate-buffered saline buffer containing 0.03% hydrogen peroxide was used as a chromogen. Images were acquired using a Zeiss Axiolab microscope  with a mechanical stage, fitted in a Sony-iris CCD video camera . The video camera was connected to a Pentium II PC, loaded with the appropriate image analysis software . Slides were examined at a ×200 magnification. The ratio, expressed in percentile proportion (%), between the number of immunohistochemically positive-stained cells and the total number (stained and unstained) of lymphocytes was calculated. All tumors had been characterized according to the following classical clinical and pathological variables: patient gender  and age , tumor location , size , grade , bowel wall invasion , lymph node metastases  and Dukes' stage . All "D" Dukes' stage cases involved hepatic metastases. The tumors were categorized according to their location into two larger groups: those involving the rectum as well as the left colon  and those involving the right colon . This was performed for reasons of statistical analysis.Moreover, positive cases were divided into two groups: those in which immunopositive cells ranged from 0–9% of the total number of lymphocytes ("weak" tissue presence of NK cells) and those in which they were ≥10% ("strong" NK cell presence). This was performed somewhat arbitrarily, as we considered that the cutoff level of 10% is in accordance to what a pathologist would consider to be the cutoff level between "negative" and "positive" cases in a qualitative examination, and in order to perform statistical analysis. Associations of NK cell presence with the patients' sex, tumor location, grade, and presence of bowel wall invasion, as well as lymph node metastases and Dukes' stage were examined using chi-square statistics; associations with the patients' age and tumor size were determined using the Mann-Whintey U test.NK cells were detectable in the primary tumor site of 79 cases (96.3%); their percentages were up to 32% of the primary site lymphocytes . Of the 33 cases with metastatic lymph nodes, NK cells were found in 27 cases (81.8%); moreover, NK cells were found among liver lymphocytes in 3 of the total 4 cases with hepatic metastases (75% of stage "D" cases).Forty-nine of the 82 primary tumors (59.8%) had a "weak" tissue presence of NK cells (0–9%), whereas the remaining 33 primary tumors (40.2%) had a "strong" NK cell presence (≥10%). Of the 33 cases with metastatic lymph nodes, 18 cases (54.5%) had "weak" and 15 "strong" NK cell presence.NK cell presence in the primary tumor site was not associated with the patients' sex , tumor size , tumor location , tumor differentiation , wall invasion , the presence of metastatic lymph nodes , or Dukes' stage . However, a negative association between the presence of NK cells at the primary tumor site with the patients' age was noticed .Moreover, the presence of NK cells in the metastatic lymph nodes was not associated with the patients' sex , age , tumor size , tumor location , tumor differentiation , wall invasion , or Dukes' stage (C or D) .Although colorectal adenocarcinoma has been characterized as a model neoplasm in which clinical prognosis is mainly determined by the histological stage, pathologic markers often fail to predict the prognosis of a great number of patients. This has necessitated the evaluation of several alternative prognostic markers . ImmunohCD16 and CD57 were chosen in the present study, as they are markers of the presence of NK cells, a cellular line which could reasonably play a significant role in the pathophysiology of colorectal adenocarcinoma. NK cells were considerably present in the primary tumor site, as well as the metastatic lymph nodes of many cases in our series. This observation is in line with data deriving from the literature -14; thisHowever, what seems to be pivotal for rapid and efficient migration of NK cells from the circulation to the tumor stroma, is the expression of appropriate adhesion molecules . As exprIn conclusion, our findings suggest that less NK cells are found in the stroma surrounding the primary tumor site in older patients with colorectal adenocarcinoma. This could possibly be attributed to decreased adhesion molecule-mediated migration; however, this hypothesis needs to be further investigated through more studies.The pre-publication history for this paper can be accessed here: