Many believe that the ethical problems of donation after cardiocirculatory death (DCD) have been "worked out" and that it is unclear why DCD should be resisted. In this paper we will argue that DCD donors may not yet be dead, and therefore that organ donation during DCD may violate the dead donor rule. We first present a description of the process of DCD and the standard ethical rationale for the practice. We then present our concerns with DCD, including the following: irreversibility of absent circulation has not occurred and the many attempts to claim it has have all failed; conflicts of interest at all steps in the DCD process, including the decision to withdraw life support before DCD, are simply unavoidable; potentially harmful premortem interventions to preserve organ utility are not justifiable, even with the help of the principle of double effect; claims that DCD conforms with the intent of the law and current accepted medical standards are misleading and inaccurate; and consensus statements by respected medical groups do not change these arguments due to their low quality including being plagued by conflict of interest. Moreover, some arguments in favor of DCD, while likely true, are "straw-man arguments," such as the great benefit of organ donation. The truth is that honesty and trustworthiness require that we face these problems instead of avoiding them. We believe that DCD is not ethically allowable because it abandons the dead donor rule, has unavoidable conflicts of interests, and implements premortem interventions which can hasten death. These important points have not been, but need to be fully disclosed to the public and incorporated into fully informed consent. These are tall orders, and require open public debate. Until this debate occurs, we call for a moratorium on the practice of DCD. There have been many "consensus" statements addressing the practice of donation after cardiocirculatory death (DCD). In general, these claim that DCD conforms to clear ethical principles, respects the dead donor rule, and is worthy of support -5. Many We argue for a moratorium on the practice of DCD until full public disclosure and fully informed consent is obtained from potential donors. Specifically, we will argue that DCD donors may not yet be dead, conflicts of interest in the decision to withdraw life support before DCD are unavoidable, potentially harmful premortem interventions during DCD cannot be justified even with the rule of double effect, consensus statements are of low quality and plagued by conflict of interest, and that claims that DCD conforms with the intent of the law and current accepted medical standards are misleading and inaccurate. Moreover, some arguments in favor of DCD, while likely true, are "straw-man arguments," such as the substantial benefit of organ donation.In general, the current practice of controlled DCD can be summarized as follows -5. FirstProtocols for DCD have attempted to clarify the justification for this practice with a series of self-evident truths -5. FirstIn discussing death it is useful to review the paradigm used to define it: death is an irreversible biological/ontological event. As Bernat has argued, death is an event separating the process of dying  from the process of disintegration -10. BernThe ordinary sense of the meaning of irreversible is "not capable of being reversed,"  and it "Suppose that Joe has a heart attack and his circulatory function stops. Fred, a physician standing next to Joe, refuses to perform CPR on Joe because Joe is a rival... Suppose that CPR would have been successful, but because it was not performed, cessation of Joe's circulatory function was permanent. Was Fred's refusal to act wrong? Not if we understand the irreversible cessation of circulatory function as equivalent to the permanent cessation of circulatory function... On that understanding, Joe was dead as soon as he collapsed, and Fred's failure to perform resuscitation was not wrong, for he had no obligation to resuscitate a corpse [p26].An objection to this scenario would be that in DCD an informed autonomous moral decision has been made not to attempt resuscitation, and this is why permanent is an acceptable 'construal' of 'irreversible' in determining death -5. This An individual is in a severe automobile accident and arrives in the ER. You are the ER physician. You judge that the patient's blood loss is so great that the patient will soon die unless she receives a blood transfusion. Her surrogates decline the transfusion because she is a Jehovah's Witness. You respect the refusal and she dies. You would say, of course: 'Her condition was reversible! I wish I could have transfused her!'... you would be wrong to say that... since reversing the patient's condition was not legally or morally permissible, the patient should have been viewed as being in an irreversible condition...[p29].is death. "To say it is makes a word mean what it does not, and to do that without warning is necessarily to mislead" .It is interesting that the proponents of DCD may in some way recognize these flaws in the declaration of death in the donor. Consensus statements simply state that 'permanent' was the 'construal' of 'irreversible' accepted by the panels -5. More We believe that these arguments may be used to make a case for organ donation in the setting of DCD; however, they cannot be used to argue that the donor is dead. What these arguments show is that the DCD donor has an almost certain prognosis of death, is in the process of dying, but is still living and not yet dead. Moreover, the accuracy of the prognosis depends on a future event , and to claim the prognosis is certain relies on backward causation. In philosophical terms, the prognosis is a "soft fact" about the past, not determined until the future events occur, and thus not a "hard fact" at all. It is a "serious logical mistake by conflating a prognosis of imminent death with a diagnosis of death" [p16]. If The consensus statements that approve of DCD were of poor quality when it comes to evidence based medicine, and based on "expert opinion" -5. It isAnother form of autoresuscitation is called the Lazarus phenomenon, which has been reported in at least 32 cases . These pThere are likely two types of Lazarus phenomenon: one occurs after a short interval of 1-2 minutes and may be due to resolution of hyperinflation with associated venous return and drug delivery to the heart; the other type with more delayed autoresuscitation cannot be so explained ,38. HypeProminent DCD proponents have claimed that determining death is not primarily an ontologic issue , nor a moral issue (whether our obligations to the patient are as if they are dead); rather, it is "fundamentally a medical practice issue" ,40p1762]p176240]pFirst, the observation that physicians often do not have a waiting time to verify irreversibility of absent circulation when commonly declaring death "is irrelevant in situations where following the dead donor rule (DDR) requires knowing whether the patient is merely dying or already dead... the accuracy that is required of our assessments depends on the consequences of our assessments being wrong... if one holds that the DDR is an inviolate principle of organ donation, then the difference between 'dying' and 'dead' becomes crucial" [p16]. Jof[the lead author of the recent Canadian forum on DCD] writes that in the past "observation and confirmation was not required and the irreversibility of death was not a practical concern, although diagnostic errors were made." Acknowledging that "diagnostic errors were made" shows that what death is has been clear in the past. Outside the context of organ donation, when one was claimed to be dead based on the irreversible loss of circulation, if the patient was subsequently revived (by autoresuscitation or intervention) and clearly alive, one simply had to admit that the pronouncement of death was incorrect. I do not believe that in this situation one would continue to insist that the diagnosis of death was correct, and that somehow the patient was revived from the irreversible state known as death. This shows that death in the past was understood as an irreversible state of dis-integration of the organism. However, in the context of DCD, we are now forced to "enhance the rigour of the determination of death," and would in this situation of revival have to explain somehow that the patient in the irreversible (or "permanent") state 'death' has now somehow become alive [p4].Accepted medical practice is to declare death when circulation is lost, and retrospectively know this was correct after a period of time that verifies it is irreversible. If this retrospective ability is taken away, then we believe physicians would be aware that death had not yet occurred when circulation stopped.was the intent seems to have evolved over time, with Bernat in earlier writings suggesting that it "may" have been the intent, and even earlier, that it was clearly not the intent .Bernat wrote in 1998:The cessation of heartbeat and breathing must be prolonged because their absence must be of sufficient duration for the brain to become diffusely infarcted and for the cessation of heartbeat and breathing conclusively to be irreversible... It takes considerably longer than a few minutes for the brain and other organs to be destroyed from cessation of circulation and lack of oxygen. Moreover, it takes longer than this time for the cessation of heartbeat and breathing to be unequivocally irreversible, a prerequisite for death. As proof of this assertion, if cardiopulmonary resuscitation were performed within a few minutes of cardiorespiratory arrest, it is likely that some of the purportedly 'dead' patients could be successfully resuscitated to spontaneous heartbeat and some intact brain function... The brief absence of heartbeat and breathing is highly predictive of death in this context [of DCD], but that at the time the organs are being procured in the Pittsburgh protocol, death has not yet occurred [p20]... the UDDA is a useful statute that has been implemented in the majority of states in the United States. Because of its unambiguous wording, succinctness, and for the sake of uniformity, I continue to support its adoption...[p21].With these writings in mind, it is remarkable the change that has occurred in more recent writings. For example, Bernat wrote that "[the 2006] national conference affirmed the ethical propriety of DCD as not violating the dead donor rule" [p281]. BeMedicine, law, ethics, the President's Commission in Defining Death, the Law Reform Commission in Canada, and proponents of DCD all agree that there is only one death per person ,41,42,50At 10 minutes of absent circulation the brain is not destroyed, and if resuscitation were implemented, some will survive with some clinical and critical brain functions -57. It iWe believe that not engaging the arguments we have made questioning some aspects of DCD has led to serious problems. Although an appeal to a utilitarian judgment is tempting, given the good consequences (beneficience) from organ donation, we believe there are also negative consequences. Alister Browne points to some "good-looking" moral principles being violated in DCD: never treat others as mere means, never interfere with the liberty of individuals when they are not doing or threatening harm to others, and never keep information concerning matters of public policy from the people in a democracy . He alsoFor proponents of DCD, the reason permanent is a surrogate for irreversible is that the team and patient/surrogate have decided that attempts to reverse it  will not be allowed ethically/legally to occur because there is an agreed upon DNR order in place. In the setting of uncontrolled DCD this has resulted in the reinstitution of CPR , or even institution of cardiopulmonary bypass , once death is declared, in order to preserve the organs -63. So, There are other reasons why our concerns with DCD are even greater in this setting of uncontrolled DCD Table . It is nQuite often, the carefully wrought initial protocols give way over time to a more 'pragmatic' approach, ultimately allowing interventions that would not have met the stringent initial conditions... Past experience demonstrates that efforts to evaluate the current protocol must anticipate that its current restrictions are likely to be relaxed significantly, here and elsewhere, once the protocol is endorsed in principle and put into practice. In my own view, approving the protocol on the basis that its current restrictive conditions will continue to provide adequate protections is an exercise in self-delusion [p229].Arnold and Youngner also warned of the "quieter strategy of policy creep" .Most DCD statements are clear that conflicts of interest shall not influence decisions -5. We beSecond, the risk of bias due to the availability of DCD cannot be simply acknowledged and stated to be unacceptable, therefore allowing one to pretend it has gone away. A Canadian multicenter study found that of 341 adult patients who were assessed by a physician on at least one occasion to have a probability of ICU survival of < 10%, 99 (29%) survived the ICU . Even foThird, proponents of DCD claim that those involved in transplantation will not be the ones who discuss DCD and obtain consent from the patient/surrogate. This is at best misleading. It may be true that the transplant surgeons will not be the ones to explain and request consent for DCD. However, it is not accurate to claim that "no physician who has had any association with a proposed transplant recipient that might influence their judgment shall take part in the determination of death" nor that "attending hospital staff caring for the recipient should be different than staff caring for the donor" [pS9]. TheFourth, we believe that conflict of interest matters are actually encouraged in order to improve adoption of DCD. The organ donation breakthrough collaborative  has been actively encouraged by proponents of DCD, including the Institute of Medicine ,77. The Although most consensus statements recognize potential conflicts of interest, we believe they make misleading claims that these conflicts are dealt with and therefore not a concern.Premortem interventions are often used to theoretically improve the condition of donated organs in DCD, although there is no evidence that they improve outcomes . These iFirst, these interventions are, in theory, to benefit the organ recipient, not the donor. It is questionable whether the donor can consent to an intervention that cannot benefit him/her and has a significant risk of harm and of hastening or causing death. It has been argued that these interventions are unlikely to hasten or cause death ,4. We beSecond, we agree with others that "to summon the protection of the principle of double effect by claiming that since donation benefits the patient and family by complying with their wishes, use of these agents is permissible, is an extreme example of professional sophistry" . In the ... the so-called 'foreseen but unintended' bad consequence in a double effect argument must be genuinely not desired... The presupposition of the conventional double effect argument is that the patient's death is the foreseen, but unintended and undesired, consequence of an effort to relieve pain... I find it simply impossible to take these assurances at face value in the context of a transaction whose entire purpose and reason for being is to bring about the death of the patient in a fashion that produces viable organs for transplantation... the protocol seeks to recharacterize a coherent, carefully worked out chain of events, leading inexorably to a foreseen, desired and planned result-death and utilization of organs for transplant- as a series of isolated links, each to be understood solely as directed to the patient's needs of the moment, each entirely disconnected from all surrounding context, including the very purpose of the exercise...[p222].after death; the family does deserve the opportunity to donate in appropriate circumstances; organ transplantation saves many lives; and people do die on transplant waiting lists. Another claim is that surveys show the public is willing to accept DCD .against DCD that we recognize also may be straw-man arguments. The process of DCD has been suggested to alter good end-of-life care. Some have called this "the forgotten donor" [There are argument n donor" . The recn donor" p82], andn donor" p86]. We n donor" ; neverthn donor" . The sugn donor" ,118,119.n donor" ,120-122.The concerns with DCD should be debated on their merits without undue deference to prominent consensus statements of expert opinion. Nevertheless, we recognize that several respected groups have published consensus statements that have been said to affirm that DCD as currently done is ethically sound -5,75,123Consensus statements in general, and those on DCD in particular, are of low quality when assessed by evidence based medicine standards . The maiThere is a large and growing list of authors who have raised many of our concerns with DCD, and who have concluded that DCD donors are not dead ,126-132.The practice of DCD has been endorsed by the American Academy of Pediatrics, including the concerning suggestion for "timely referral...[that] may start in the emergency department with the admission of a critically injured child... Deliberation with the OPO should occur before or when...'withdrawal-of-care' or 'do-not-resuscitate' options are being discussed" [p823]. OfConcerns with DCD are increased in children. First, there is even less data regarding autoresuscitation and Lazarus phenomenon; only 6 children were included in the studies identified by the Institute of Medicine Table  19-21],-21,19-21In children (particularly under age 14 years), premortem decisions are made using the best interests standard by surrogate decision makers, usually the parents ,140. ThiThese concerns are not merely theoretical. A recent review of pediatric DCD policies found variable and concerning practices Table  142]. S. S142]. That DCD violates the dead donor rule leads to important implications. If the dead donor rule is inviolable, then we must change the practice of organ donation to make it truly consistent with the dead donor rule, risking the lower quality of organs that would be donated. If the dead donor rule is not inviolable, then informed voluntary consent in terminally ill patients to violate the dead donor rule and allow organ donation as the proximate cause of death is required ,127,143.We believe that truthful, complete, voluntary, fully informed consent to organ donation is required. This best respects patient autonomy ,140,147.deceased donor from donation, and the decision may occur completely outside the doctor-patient relationship (as in signing a donor card or onto a donor registry) [premortem interventions are not done after death, and have real harms and benefits that require informed consideration by potential donors. In addition, the end-of-life (prior to death) care provided to the donor is altered and should require informed consent [A potential challenge to our call for fully informed consent could be the claim that organs are property, and organ donation is governed by gift law. It has been argued that organ donation would thus require only a donation intent, and not informed consent, as there are neither risks nor benefits to a egistry) -151. Thiegistry) ,153. We egistry) ,155. Secegistry) . Third,  consent . Fourth, consent ,154,156, consent  is possiThe United States Center for Medicare and Medicaid Services has held that "... there must be a minimum standard to assure that when families provide consent, they are providing informed consent... potential donor families receive the information they need to make an informed decision about donation..." . A largeWe have argued that DCD donors are not dead, and therefore that organ donation during DCD violates the dead donor rule. Our concerns with DCD include the following: irreversibility of absent circulation has not occurred and the many attempts to claim it has all fail; conflicts of interest at all steps in the DCD process are simply unavoidable; premortem interventions to preserve organ utility are not justifiable; and consensus statements by respected medical groups do not change these arguments. The truth, we believe, is that honesty requires that we face these problems instead of avoiding them. Until the concerns we describe are seriously considered, full public disclosure occurs, and fully informed consent is obtained from donors, there should be a moratorium on the practice of DCD. We believe that DCD is not ethically allowable because it abandons the dead donor rule, has unavoidable conflicts of interests, and implements premortem interventions which can hasten death. These important points have not been, but need to be fully disclosed to the public and incorporated into fully informed consent. These are tall orders, and require open public debate. Until this debate occurs, we call for a moratorium on the practice of DCD.The authors declare that they have no competing interests.ARJ drafted the first version of the manuscript. All authors made substantial contributions to conception and design of the manuscript, revising the manuscript critically for important intellectual content, and have read and approved the final manuscript. Each author participated sufficiently to take public responsibility for the content. There was no funding for this manuscript.


The published literature regarding the relationships between retinol-binding protein 4 (RBP4) and cardiometabolic risk factors and subclinical atherosclerosis is conflicting, likely due, in part, to limitations of frequently used RBP4 assays. Prior large studies have not utilized the gold-standard western blot analysis of RBP4 levels.Full-length serum RBP4 levels were measured by western blot in 709 postmenopausal women screened for the Kronos Early Estrogen Prevention Study. Cross-sectional analyses related RBP4 levels to cardiometabolic risk factors, carotid artery intima-media thickness (CIMT), and coronary artery calcification (CAC).P = 0.01), but were unrelated to blood pressure, cholesterol, C-reactive protein, glucose, insulin, and CIMT levels . Results suggested a curvilinear association between RBP4 levels and CAC, with women in the bottom and upper quartiles of RBP4 having higher odds of CAC . However, a squared RBP4 term in regression modeling was non-significant (P = 0.10).The mean age of women was 52.9 (± 2.6) years, and the median RBP4 level was 49.0 (interquartile range 36.9-61.5) μg/mL. Higher RBP4 levels were weakly associated with higher triglycerides (age, race, and smoking-adjusted partial Spearman correlation coefficient = 0.10; In these healthy, recently postmenopausal women, higher RBP4 levels were weakly associated with elevations in triglycerides and with CAC, but not with other risk factors or CIMT. These data using the gold standard of RBP4 methodology only weakly support the possibility that perturbations in RBP4 homeostasis may be an additional risk factor for subclinical coronary atherosclerosis.ClinicalTrials.gov number NCT00154180 Obesity, a worldwide epidemic, is associated with a dramatic increase in the prevalence of type 2 diabetes, and has been associated with cardiovascular disease (CVD) morbidity and mortality -3. RetinThe purpose of the current study was to examine the associations of RBP4 with both standard CVD risk factors as well as subclinical atherosclerosis in healthy mid-life women screened for the Kronos Early Estrogen Prevention Study (KEEPS). Western blot, the gold standard analytic technique for RBP4 , was use2, LDL cholesterol > 190 mg/dL or triglycerides >400 mg/dL, diabetes, uncontrolled hypertension, or with moderate subclinical CVD, defined as a coronary artery calcium (CAC) score ≥50 Agatston units, were ineligible for randomization. Women meeting eligibility criteria were randomized to one of three treatment groups: oral conjugated equine estrogen  with oral micronized progesterone (12 days per month), transdermal 17β-estradiol  with oral micronized progesterone (12 days per month), and one daily placebo group (inactive pill/patch) with placebo micronized progesterone (12 days per month). The current analyses utilize data from the screening and baseline KEEPS visit, prior to randomization and receipt of any estrogen therapy or placebo. A total of 1,046 women were screened between July 2005 to June 2008, with 775 of them passing earlier screening components and eventually receiving a CT scan for the purposes of confirming CAC Agatston score eligibility. Of these, 66 women were missing RBP4 data, leaving 709 women for the current analyses. The institutional review boards of the participating institutions approved this study, and all women signed informed consent.The current cross-sectional analyses utilize data from the KEEPS, a randomized, placebo-controlled, double-blinded, prospective trial of the effects of menopausal hormone therapy on the progression of subclinical atherosclerosis in recently menopausal women. KEEPS is a multi-center trial with 9 centers . KEEPS inclusion criteria were age 42–58 years, at least 6 months but no more than 36 months of amenorrhea and last spontaneous menses occurring after age 40, FSH values ≥35 ng/ml and/or estradiol levels <35 pg/ml, and good general health. Women reporting use of estrogen- or progestin-containing medication , selective estrogen receptor modulators (SERMs), or medications with significant estrogenic activity, such as isoflavones, in the prior 3 months, a history of clinical CVD, current heavy smoking , a body mass index (BMI) ≥35 kg/mAll blood specimens were collected following an overnight fast, then frozen at −70°C on site until they were either processed locally, or sent to the Kronos Science Laboratory  for storage or assays. Complete blood count and chemistry panel were performed at the clinical laboratories at each recruiting center. C-reactive protein (CRP) and insulin were assayed by DPC Immulite 2000 .RBP4 in human serum was measured by quantitative western blotting . BrieflyBaseline smoking status (current use yes/no) and race-ethnicity were obtained through self-reported questionnaire. Height (cm) and weight (kg) of participants, wearing light clothing and without shoes, was measured with stadiometers and calibrated balance-beam scales, respectively. BMI was calculated as weight in kilograms divided by the square of height in meters. Waist circumference (cm) was measured with a non-stretchable tape at the end of a normal expiration, at the smallest horizontal circumference between the ribs and iliac crest. Blood pressure was measured in the right arm in the seated position, after at least a 5 minute rest, and was averaged across two readings.Carotid artery intima-media thickness (CIMT) was measured from high resolution B-mode ultrasound images obtained with a 7.5 MHz transducer at both the screening and baseline KEEPS visits. Carotid artery images were acquired at each study center by certified ultrasound technicians trained at the core CIMT imaging and reading center  to perform standardized image acquisition. Image acquisition was optimized for minimal measurement variability (coefficient of variation <2%) using methodology developed specifically for reproducible longitudinal imaging employing internal anatomical structural probe angulation techniques including vessel stacking, baseline image matching, electrocardiographic gating, and individualized instrumentation conditions  . All ima2) with a density of >130 Hounsfield units. The individual lesion score was calculated by multiplying the lesion area by a density factor derived from the maximal Hounsfield unit within this area, as described by Agatston et al. [Each participant underwent non-enhanced cardiac computed tomography (CT) scans for assessment of coronary atherosclerosis. Scans were acquired either via electron beam CT , or via multidetector CT using either a General Electric helical scanner or a Siemens or Phillips multi-slice scanner (minimum requirement 4 detector heads). Comparability among centers was assured by regular calibration using a standard phantom. The scans were obtained during a single breath hold at end inspiration to reduce motion artifacts and improve image quality of the coronary arteries. Coronary calcium was defined as a plaque of at least 3 contiguous pixels , median (interquartile range), or n (%) were tabulated for demographic, behavioral, and laboratory characteristics by quartiles of RBP4. Differences in the distributions of these variables across RBP4 quartiles were calculated by the Chi-Square test for categorical variables, ANOVA for normally distributed continuous variables, and the Kruskal-Wallis test for non-normally distributed continuous variables. Following this, partial Spearman correlation coefficients were calculated for associations between RBP4 and cardiometabolic risk factors and CIMT, adjusted for age, race (white vs. non-white), and current smoking. To assess associations between RBP4 and CAC, multivariable adjusted logistic regression was used to calculate odds ratios of CAC score >0 across quartiles of RBP4, as well as treating RBP4 as a continuous variable after log-transformation, adjusted for age, race, and current smoking, and again after additional adjustment for triglycerides. To ensure that residual confounding by smoking was not influencing associations, sensitivity analyses were carried out excluding current smokers from regression analyses.P = 0.01), the associations of cardiometabolic risk factors and RBP4 remained null  after multivariable adjustment for age, race-ethnicity, and smoking status . To further explore the possibility of a curvilinear association, multivariable-adjusted logistic regression models for CAC were initially performed with the 1st RBP4 quartile as the reference category  after adjustment for age, race, and current smoking (Tablerd and 4th RBP4 quartiles were not significantly associated with CAC. When using the 2nd RBP4 quartile as the reference category (37.0-49.0 mg/mL) . Due to the curvilinearity suggested by the RBP4-CAC relationship, logistic regression analyses tabulating odds ratios of the top tertile of each cardiometabolic risk factor associated with being in the 2nd RBP4 quartile vs. the 1st, 3rd, and 4th were carried out similar to Model 2 in TableThe mean age of study participants was 52.9 (± 2.6) years. Demographic and laboratory characteristics stratified by quartiles of RBP4 are presented in TableAmong these healthy, recently postmenopausal women, higher RBP4 levels were weakly associated with greater triglyceride levels, and both low and high RBP4 levels were associated with approximately twice the odds of the presence of CAC. No associations were observed between RBP4 levels and other cardiometabolic risk factors or CIMT.RBP4 has long-been known to be released by the liver, but recently, it has been shown that approximately 15% of circulating RBP4 levels results from adipose tissue RBP4 secretion . RBP4 doP < 0.001) [P < 0.001) but not with other cardiometabolic risk factors [Retinoids have been known to affect the expression of several genes involved in triglyceride metabolism, including regulators of apo C-III production, hepatic and gastrointestinal triglyceride production and secretion, and beta-oxidation of fatty acids . As such< 0.001) . A study factors . The preThe association between RBP4 and CIMT, a measure of systemic atherosclerosis, has been investigated in only a few studies, and as with relationships between RBP4 levels and obesity, insulin resistance, and other CVD risk markers, results conflict. Despite recent data suggesting that increases in fasting plasma glucose even within the normal range are associated with worse arterial stiffness levels , reportend RBP4 quartile) RBP4 levels. The odds of having a CAC score > 0 in women in either the 1st or 3rd quartiles of RBP4 were approximately 2 times higher than that among women with RBP4 levels in the 2nd quartile. Similarly, women in the 4th RBP4 quartile had a non-significantly 64% higher odds of having a CAC score > 0 compared to women in the 2nd RBP4 quartile. To our knowledge, this is the first investigation to assess the association of RBP4 with the presence of CAC. These findings require replication in future studies and need to be interpreted with considerable caution given the lack of a significant squared RBP4 term in regression analyses. Caution is especially indicated given the relatively small number of women with CAC score > 0 in the current study , and the null association with CIMT, an alternative measure of atherosclerosis. Had RBP4 levels been associated with measures of glucose homeostasis as in certain prior studies, a u-shaped association with coronary atherosclerosis might be supported given the heightened CVD death not only with hyperglycemia, but also potentially with hypoglycemia, as suggested by the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial results [In contrast to our findings with CIMT and to earlier findings noted above with ankle-brachial index, we observed a significantly higher odds of the presence of CAC in women with both low and high RBP4 levels compared to women with moderate  but a null association with RBP4 , suggesting that null associations between RBP4 and HDL and glucose were not a result of the measurement schedule.Some limitations of this study must be taken into consideration. The design of this study was cross-sectional, so the results do not imply causality. Our study sample also included only healthy, recently menopausal women. Therefore, our results may not be applicable to an older population with greater prevalence of atherosclerosis. Additionally, glucose and lipids were obtained from blood collected during screening, while insulin, CRP and RBP4 were obtained from blood collected at the baseline randomization examination. Therefore, we were unable to calculate insulin resistance via the homeostasis model assessment (HOMA-IR), and a bias towards the null was introduced for associations between RBP4 and lipids and glucose. However, insulin levels, despite being measured at the same alternate visit as RBP4, showed moderate to strong associations with lipids and glucose (Spearman correlation coefficient of −0.4 with HDL, 0.4 with triglycerides, and 0.3 with glucose; all The primary strength of this study involves the method of RBP4 detection. The quantitative western blot methodology that we have chosen has been cited as the gold-standard for RBP4 determination , and utiThese analyses in healthy, recently postmenopausal women suggest that higher RBP4 levels are weakly associated with elevations in triglycerides, and that both low and high RBP4 levels may be associated with subclinical coronary atherosclerosis. Given the caution with which the coronary atherosclerosis results should be viewed, our current results in healthy midlife women only weakly support a role for RBP4 homeostasis in cardiometabolic abnormalities and cardiovascular disease. However, future studies should attempt to replicate our CAC findings. If successful, RBP4 may end up warranting examination as a potential target of interventions to reduce CVD risk.ACCORD: Action to control cardiovascular risk in diabetes; BMI: Body mass index; CAC: Coronary artery calcification; CIMT: Carotid intima media thickness; CRP: C-reactive protein; CT: Computed tomography; CV: Coefficient of variation; CVD: Cardiovascular disease; GLUT4: Glucose transporter type 4; KEEPS: Kronos Early Estrogen Prevention Study; LDL: Low-density lipoprotein.The authors have no competing interests to disclose.GH wrote the manuscript; RPW conceptualized the research hypothesis and analyses, assisted in writing the manuscript, and edited the manuscript; DW performed all of the statistical analyses; JEM and NS assisted in conceptualizing the research question and reviewed and edited the manuscript, MJB, HNH, MSH, and YS researched data, and reviewed and edited the manuscript, EAB, MIC, UIK, RAL, GRM, VM, FN, HST, and IZ reviewed and edited the manuscript. All authors read and approved the final manuscript.


Borrelia burgdorferi, HIV also has neurotropic properties. Elevated serum CXCL13 concentrations have been reported in HIV patients, but data on CSF levels are limited.It has been suggested that cerebrospinal fluid (CSF) CXCL13 is a diagnostic marker of Lyme neuroborreliosis (LNB), as its levels have been shown to be significantly higher in LNB than in several other CNS infections. Levels have also been shown to decline after treatment with intravenous ceftriaxone, but levels after treatment with oral doxycycline have previously not been studied. Like We longitudinally analysed CSF CXCL13 concentrations in 25 LNB patients before and after oral doxycycline treatment. Furthermore, we analysed CSF CXCL13 concentrations in 16 untreated LNB patients, 27 asymptomatic untreated HIV-1 infected patients and 39 controls with no signs of infectious or inflammatory disease.In the longitudinal LNB study, initially high CSF CXCL13 levels declined significantly after doxycycline treatment, which correlated to a decreased CSF mononuclear cell count. In the cross-sectional study, all the LNB patients had CSF CXCL13 levels elevated above the lowest standard point of the assay (7.8 pg/mL), with a median concentration of 500 pg/mL . Of the HIV patients, 52% had elevated CSF CXCL13 levels . There was a clear overlap in CSF CXCL13 concentrations between LNB patients and asymptomatic HIV patients. All but one of the 39 controls had CSF CXCL13 levels below 7.8 pg/mL.We confirm previous reports of highly elevated CSF CXCL13 levels in LNB patients and that these levels decline after oral doxycycline treatment. The same pattern is seen for CSF mononuclear cells. CSF CXCL13 levels are elevated in neurologically asymptomatic HIV patients and the levels overlap those of LNB patients. The diagnostic value of CSF CXCL13 in LNB remains to be established. Borrelia burgdorferi (Bb) antibody production [Lyme neuroborreliosis (LNB) is the most common arthropod-borne CNS infection in Europe and the USA . The diaoduction ,3. Thereoduction ,5.In recent years, the chemokine CXCL13  in CSF has been presented as a potential marker of LNB, more specific than today’s diagnostic approach . CXCL13 Oral doxycycline and intravenous ceftriaxone are considered equally effective for the treatment of LNB with peripheral nervous system symptoms, while intravenous ceftriaxone is still the preferred treatment of LNB with central nervous system symptoms such as meningitis or encephalitis in many centra .HIV infects the central nervous system at the primary infection or shortly thereafter, causing a chronic, low-grade inflammatory reaction in most clinically asymptomatic patients ,12. In HWe analysed CSF levels of CXCL13 in patients with LNB, HIV and controls, in order to assess whether the test is specific for LNB even when compared with patients with HIV infection. We have also studied how doxycycline treatment of LNB affects CSF CXCL13 levels and the CSF cell count by analysing a well-characterised set of LNB patients before and after treatment.We enrolled different study participants for the longitudinal and the cross-sectional study. For the longitudinal study, we analysed 25 LNB patients who had undergone CSF sampling before and after treatment between October 1995 and November 2005. All these patients were treated with 200–400 mg of oral doxycycline daily for 10–14 days, according to Swedish recommendations . In accoBb-specific antibodies in CSF above the upper reference level; IV. At least one of the following: a) a positive antibody index (AI), defined as the ratio of the CSF/serum quotient of specific Bb antibodies to the corresponding CSF/serum quotient of total immunoglobulins, where AI values of ≥ 1.5 are considered positive, b) CSF cytological examination consistent with LNB with activated plasma cells [The diagnostic criteria for LNB were: I. Symptoms consistent with LNB and other diagnoses excluded; II. CSF mononuclear pleocytosis with a mononuclear cell count of > 5/μL and erythrocytes of < 100/μL; III. ma cells .All CSF samples were collected by lumbar puncture in the L3/L4 or the L4/L5 inter-space. The first 5–12 mL of CSF were collected in a polypropylene tube and immediately transported to the local laboratory for centrifugation. The supernatant was pipetted off, gently mixed to avoid possible gradient effects and aliquoted in polypropylene tubes that were stored at −80°C pending biochemical analyses, without being thawed and re-frozen.Bb antibodies in serum and CSF, two different tests were used during the study period. Until 26 June 2006, antibodies were analysed using an enzyme-linked immunosorbent assay (ELISA) kit for IgG and IgM antibodies . Tests positive for IgM were further analysed with a more specific test . After 26 June 2006, Bb antibodies were analysed using a sandwich chemiluminescence immunoassay (CLIA) test kit . HIV RNA in serum and CSF was determined using a quantitative polymerase chain reaction .CXCL13 was measured by ELISA , according to instructions from the manufacturer. Based on measurements of duplicates of the standard samples (concentrations 7.8-500 mg/L), the average intra-assay CVs were ≤ 10%. Syphilis testing was done with LIAISON Treponema Screen . For the analysis of Statistical analysis was performed using GraphPad Prism 5.0 . Data are presented as the median (range). CXCL13 values below the detection limit of 7.8 ng/mL were assigned a value of 3.9 ng/mL for graphical purposes. Analyses were made using non-parametric methods. In the longitudinal study, differences before and after treatment were analysed with the Wilcoxon matched pairs test. Differences between groups in the cross-sectional study were analysed with the Kruskal-Wallis test followed by Dunn’s post test. Correlations were analysed using the Spearman rank order correlation. P values of < 0.05 were considered significant.In the longitudinal part of the study, 25 LNB patients were analysed. Baseline data, clinical symptoms and routine CSF analyses are shown in Table In the cross-sectional part of the study, 85 patients were analysed; 16 with LNB, 27 with HIV infection and 39 controls without inflammatory CNS disease. Baseline data, clinical symptoms and routine CSF analyses are shown in Table CSF CXCL13 levels differed significantly between all three groups of patients in the cross-sectional study  but the difference was not statistically significant (P = 0.065).r = 0.81, P < 0.001) , and in the pre-treatment part of the longitudinal study . The combined analysis of the two groups of untreated LNB patients produced a Spearman r = 0.55, (P < 0.001)  Figure . For the) Figure .For LNB patients, there was no significant correlation between the duration of neurological symptoms and CSF CXCL13 levels, either in the longitudinal study or in the cross-sectional study (data not shown).For the assessment of the diagnostic performance of CSF CXCL13, we combined the data on LNB patients from the cross-sectional study and the pre-treatment part of the longitudinal study and analysed them against the combined group of HIV patients and controls. A receiver operating characteristic (ROC) curve is shown in Figure We confirm previous findings that CSF CXCL13 levels are highly elevated in untreated LNB patients. We also show that CSF CXCL13 concentrations decline after treatment with oral doxycycline and that the levels before and after treatment differ by about a hundredfold. This finding supports to the use of oral doxycycline for the treatment of LNB. Oral treatment has the advantages of being both cheaper and more convenient for the patient compared with intravenous treatment.Bb-specific antibody production can be seen in patients during the first few weeks of disease and, in a small number of patients, CSF pleocytosis may be absent for the first days of disease [Does CSF CXCL13 concentrations add any valuable information to routine CSF analysis? Initially elevated CSF mononuclear cell count also declines significantly after treatment and the decline in levels of CSF mononuclear cells and CXCL13 are correlated, as shown in Figure  disease ,18. In t disease . A prospIn the cross-sectional study, we chose to compare the levels of CSF CXCL13 LNB patients with patients with HIV infection for two reasons. Elevated serum CXCL13 levels have been described in HIV patients. Serum CXCL13 levels were higher in HIV patients with more advanced disease and correlated moderately with viral load . ElevateThe mechanism behind the previously reported elevated serum CXCL13 concentrations in HIV patients has not been determined. CXCL13 is known to be produced by macrophages, but the direct stimulation of macrophages and peripheral blood mononuclear cells by HIV-1 failed to induce CXCL13 production in one study . In thisIt is difficult to compare CSF CXCL13 results from various studies. CSF CXCL13 has previously been related to CSF protein  to correlate with impaired blood brain barrier function and possible leakage into the CSF of CXCL13 . The facPrevious studies of CSF CXCL13 in LNB have suggested optimal cut-off values, maximising sensitivity and specificity, where the level of CSF CXCL13 should be considered positive for LNB. Proposed cut-offs range from 142 pg/mL to 1,229 pg/mL  ,23,25. WWe confirm previous reports of highly elevated CSF CXCL13 levels in LNB patients. CSF CXCL13 concentrations decline a hundredfold after treatment with oral doxycycline, which support the efficacy of this regimen. The same pattern is seen for CSF mononuclear cells. We show that CSF CXCL13 levels are also elevated in neurologically asymptomatic HIV patients and that the levels overlap those of LNB patients. CSF CXCL13 levels in LNB patients differ widely between studies. The diagnostic value of CSF CXCL13 in LNB remains to be established.NM has served on a scientific advisory board for Actelion Inc. LH has participated in a scientific advisory board for Pfizer, Astra Zeneca and Meda. The other authors report no competing interests.DB, NM, HZ and LH came up with the idea for this study and participated in its conception and design. DB and LH drafted the manuscript. KB and UA participated in setting up the analyses. DB, ME, CW and LH recruited the study participants. NM and HZ made important revisions to the manuscript. All the authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2377/13/2/prepub


Intratesticular arteriovenous malformations (AVMs) are extremely rare benign incidental lesions of the testis. Ultrasonography (US) generally reveals a hypoechoic solid mass within the testicular parenchyma. We describe a patient with intratesticular AVM which was found incidentally during workup for infertility. The gray-scale and Doppler US appearance of an intratesticular AVM and the differential diagnosis have been presented. Based on the gray-scale, US appearance differentiation from malignant testicular tumors is difficult. Doppler US examination aids in the diagnosis by demonstrating the vascular nature of the tumor. Testicular cancer is the most common cancer in men aged 20–34 years and accounts for 1% of all malignancies in men. Gray-scale ultrasound (US) is the standard imaging technique used to identify testicular carcinoma and nearly 100% sensitive for detection of testicular tumors [α-fetoprotein and human chorionic gonadotropin levels were within normal limits. Gray-scale images showed a well circumscribed, 5.7 mm sized hypoechoic round lesion within the left testis . FSH level was in the upper limit of normal. The t testis . Color Dt testis . There wt testis . The rest testis  on spectAVMs involving the lower urinary tract are uncommon as opposed to AVMs located in the Central Nervous System. Arteriovenous malformations of the spermatic cord and testis are benign lesions consisting of complex tangles of enlarged dilated arteries and veins without intervening capillaries. They can present as either painless paratesticular masses or as incidental findings during evaluation for infertility or as combination of both infertility and scrotal swelling and rarely as recurrent acute scrotal pain .Most intratesticular masses are malignant and ultrasonography is the method of choice in the determination of the mass nature (cystic or solid), size, and vasculature. Besides primary testicular tumors a number of cystic appearing benign lesions such as testicular cysts, tubular ectasia of the rete testis, intratesticular varicocele, and solid-appearing benign lesions such as lipomas, hemangiomas, and AVMs can be observed , 4, 5. IOf the cystic lesions, testicular cysts appear with an anechoic center, through-transmission, and without a perceptible wall . There iUse of color and power Doppler US in malignant tumors of the testis help to better define testicular involvement . The preMagnetic resonance (MR) imaging is an important imaging technique in the evaluation of scrotal masses, providing a useful adjunct to ultrasonography (US). MR imaging allows tissue characterization, with its signal intensity properties allowing detection of fat, blood products, granulomatous tissue, and fibrosis. MR imaging performed after intravenous administration of gadolinium-based contrast material allows more accurate assessment of the vascularity of testicular lesions than color Doppler US does. The pattern of enhancement of scrotal lesions can also be evaluated. MR can also help localize the lesion as intra- or extratesticular and can clearly identify an undescended testis. The main differential diagnosis of an AVM, if all other possibilities have been excluded, is that of a hemangioma. The MRI appearance of hemangioma is poorly documented. Essig et al. reported the MRI findings of a capillary hemangioma in a 26-year-old man . The tumIn conclusion, intratesticular AVMs are extremely rare lesions of the testis with solid and hypoechoic appearance on gray-scale US resembling a malignant neoplasm. Color Doppler US aids in the diagnosis by showing the vascular nature of the lesion and the prominent venous drainage.


Research on the evolution of signaling has shown that animals frequently alter visual features, including color cues, to attract, intimidate or protect themselves from conspecifics. Humans engage in conscious manipulation of visual signals using cultural tools in real time rather than genetic changes over evolutionary time. Here, we investigate one tool, the use of color cosmetics. In two studies, we asked viewers to rate the same female faces with or without color cosmetics, and we varied the style of makeup from minimal , to moderate , to dramatic (glamorous). Each look provided increasing luminance contrast between the facial features and surrounding skin. Faces were shown for 250 ms or for unlimited inspection time, and subjects rated them for attractiveness, competence, likeability and trustworthiness. At 250 ms, cosmetics had significant positive effects on all outcomes. Length of inspection time did not change the effect for competence or attractiveness. However, with longer inspection time, the effect of cosmetics on likability and trust varied by specific makeup looks, indicating that cosmetics could impact automatic and deliberative judgments differently. The results suggest that cosmetics can create supernormal facial stimuli, and that one way they may do so is by exaggerating cues to sexual dimorphism. Our results provide evidence that judgments of facial trustworthiness and attractiveness are at least partially separable, that beauty has a significant positive effect on judgment of competence, a universal dimension of social cognition, but has a more nuanced effect on the other universal dimension of social warmth, and that the extended phenotype significantly influences perception of biologically important signals at first glance and at longer inspection.Research on the perception of faces has focused on the size, shape, and configuration of inherited features or the  First impressions based on facial appearance occur automatically, are difficult to overcome, and impact decision-making. The strong motivational influence of facial beauty has been shown in studies of labor markets suggesting that there is a ”beauty premium” and “plainness penalty” Darwinian approaches posit that features of beautiful faces are important biological signals of mate value that motivate behavior in others, and have identified features such as averageness, symmetry and sexual dimorphism as key contributors to female facial beauty biological phenotype, such as the shape, size, configuration and movement of facial features, and ignored the effects of facial adornment and grooming, or “the extended phenotype.” The extended phenotype To date, face research has focused largely on the Research on the evolution of signaling has shown that animals frequently alter or exaggerate visual features, including color cues, to attract, mimic, intimidate or protect themselves from conspecifics, sometimes setting off an arms race between deception and the detection of such deception Experimental studies by Tinbergen first showed that it was possible to exaggerate a visual sign stimulus and produce a super-normal stimulus that elicits a super-normal response Here we examine one example of the human extended phenotype, the use of color cosmetics, a tool used primarily by women to enhance facial attractiveness. The use of cosmetics is ancient. Analyses of Egyptian cosmetic powders dated from 1200 to 200 BC show that very sophisticated wet chemical technology was already being used in their creation As popular agents of self-advertising, cosmetics have been subject to shifting cultural attitudes toward their use. They were apparently considered so good at deceiving husbands In the late eighteenth century, and so feared by them, that the English government proposed a law stating that, “All women…that shall from and after this act impose upon, seduce or betray into matrimony any of his Majesty's subjects by the use of scents, paints, cosmetics, washes, … shall incur the penalty of the law now in force against witch craft and like misdemeanors and that the marriage upon convictions shall stand null and void” In current culture, cosmetics are seen as freely chosen and morally neutral agents of beauty enhancement. Their use reflects the individual's preferences and choices, and the response to their use reflects the perceiver's attitudes about forms of self-presentation and grooming practices. Thus, when viewing a face with makeup, perceivers make inferences based not only on cosmetics' effects on the appearance of symmetry, clearness of skin or featural contrast, but on their conscious ideas about makeup use and what it may signify about the user's personality, character, and intentions.We hypothesize that cosmetics will impact face perception at a system 1 and system 2 levels, engaging both a reflexive and a reflective response. We predict that at an automatic, implicit level, cosmetics will have uniformly positive effects on judgments of beauty, personality and character. We predict that with longer inspection time, cosmetics will continue to enhance attractiveness, but may no longer uniformly enhance judgments of likability or trust, given different social attitudes toward cosmetics use. On longer inspection we expect to find greater beauty, but not necessarily the halo surrounding it for all looks.Finally, to measure one potential source of the cosmetic effect on face perception we measured luminance contrast between the eyes, lips, and the surrounding skin for faces without makeup and for each of the makeup looks. Russell We have obtained ethics approval for our study from the Partners Human Research Committee at Massachusetts General Hospital, where participants were recruited and human experimentation was conducted. This project meets the criteria for exemption from further IRB review per the regulations found at 45 CFR 46.101(b) (2) Use of educational tests , survey procedures, interview procedures, or observation of public behavior. Instead of a consent form, we were instructed to provide subjects with a fact sheet and not collect a signature. The individuals/subjects pictured in this manuscript have given written informed consent to publication of their case details. By signing our consent form and photo release, they permit us to publish or make other public use of their facial digital images with the understanding that their names and/or personal information will not be made public.To test the impact of cosmetics on judgments of faces we conducted two studies in which the same models were judged with and without makeup.Subjects in the first study included 149 adults  of different ethnicities who were shown the faces for 250 ms. The second study included 119 adults  of different ethnicities given unlimited time to inspect each face.The 100 high-resolution color images were of 25 women ages 20–50, self-identified as Hispanic, Caucasian, or African American shown in a frontal headshot with a neutral facial expression, and cropped to remove clothing and hairstyle. We used female models only, as they represent the vast majority of facial cosmetic users. A professional photographer took the images under uniform conditions. Makeup was applied by a makeup artist and then adjusted digitally. Each model was photographed without makeup and with 3 makeup looks ranging from minimal to moderate to dramatic, looks we informally labeled “natural”, “professional” or “glamorous” . We neveSubjects were presented the stimuli on an iMac desktop. Each subject saw all four looks for each of the 25 models in a randomized sequence and rated them for attractiveness, likeability, trustworthiness, and competence using a 7-point “slider scale” ranging from end points labeled “not at all” to “highly/extremely.”Our statistical analyses of makeup by inspection time were obtained from a linear mixed effects model with crossed random effects, similar to the model described by Baayen et al. Overall, makeup  produced a significant positive main effect on judgments of all outcomes in the 250 ms presentation  and on judgments of attractiveness, p<0.0001), competence (p<0.0001), and likeability (p<0.0001) , but not trustworthiness on longer inspection (p = 0.8988) .Analysis of the effects of the individual makeup “looks” revealed that each had a significant positive effect on judgments of competence and attractiveness both at 250 ms and at longer inspection times . With unf  =  (feature luminance – skin luminance)/(feature luminance + skin luminance) Finally, to determine luminosity contrasts, color images were individually hand-labeled to define 4 regions: the eyes, including the eye itself , eyelashes, the skin between the epicanthal fold and the eye; and the skin immediately below the eye; the lips; the annuli surrounding the eyes; and an annulus surrounding the lips. Then using a Spectra® PhoRad® Photometer, luminance values in candelas per meter squared (cd/m2) were collected from the iMac computer screen within the four regions and averaged, yielding mean luminance values for each of the four regions. The mean luminance for the eyes and lips were averaged to produce the mean feature luminance, and the mean luminance values for the eye and lip annuli were averaged to produce the mean skin luminance. Facial contrast was calculated as CAnalyses confirmed that each pair of looks was significantly different in level of facial luminance contrast |≥5.87, p<.0001, Bonferroni corrected significance level  = 0.05/18 = 0.003), except the natural look versus no makeup, which was marginally non-significant (t(72) = -1.83, p = 0.0717)  and S2. In summary, we found evidence of significant effects of cosmetics in both studies. All the makeup looks significantly increased attractiveness and competence ratings at 250 ms and on longer inspection. Ratings of likeability and trust varied with makeup look and inspection time, suggesting that they elicited different reflexive and reflective responses.Our results have a number of implications. As predicted, makeup had significant positive effects on ratings of female facial attractiveness at brief and longer inspection times. Ratings of competence increased significantly with makeup look tested on first glance and longer inspection. Effects were weaker and more variable for ratings of likability and trustworthiness, although generally positive.Social psychologists have suggested that social warmth and social competence represent two universal dimensions of social perception by which we evaluate individuals and groups, Past studies have shown that attractive people are expected to do better on the job, in school, and in life – and are treated that way – by being agreed with, deferred to, helped, and granted larger personal space But, as sociologists Webster and Driskell noted when first proposing the idea of beauty as status, there are important differences between attractiveness and other status characteristics such as race or sex: beauty is a malleable characteristic The beauty halo effect has been called the “what is beautiful is good” effect. In our study, makeup increased inferences of warmth and cooperation (likability and trustworthiness) when faces were presented very briefly, but did not always do so on longer inspection. In general, there is less agreement about whether beauty invariably signals social cooperation, with some studies suggesting that there is a ”dark side” to beauty characterized by vanity, immodesty, or greater likelihood to cheat on a partner Our study looked at one potential source of the cosmetics effect on face perception, increasing luminance contrast between the features (eyes and lips) and the surrounding skin, and looked for the first time at luminance contrast in African American and Hispanic faces. We found that cosmetics increased luminance contrast by significantly darkening the eyes and lips. Skin was neither significantly lightened nor darkened. However, luminance contrast effects for our natural look compared to a face without makeup was only marginally significant. It is likely that cosmetics induced image changes other than changes in luminance contrast contributed to our effects. These include possible changes in the smoothness of skin tone, in the redness of skin color or lip color, and in shading that accentuates the cheekbones. Previous research has shown that makeup can improve skin appearance, evenness, and texture to appear healthier, fertile, and youthful Finally, our study included only North American subjects; we do not know if such effects will be found in subjects from other cultures.In sum, we show that faces with cosmetics engage both fast, reflexive processes, and more deliberative conscious processes. The fast, automatic effects are uniformly strong and positive for all outcomes. In situations where a perceiver is under a high cognitive load or under time pressure, he or she is more likely to rely on such automatic judgments for decision-making. Facial images appear on ballots, job applications, websites and dating sites. Our results underscore the malleability of judgments derived from facial images of a single individual at zero acquaintance, judgments that can be highly consequential. When inferring trustworthiness, likeability, or competence from an image, we are influenced significantly not only by the attractiveness of the inherited phenotype but by the effects of the “extended phenotype,” in this case, makeup.Table S1Comparisons of mean luminosity measures for the different makeup looks obtained from the luminosity analysis regression models see .(DOCX)Click here for additional data file.Table S2Regression models for the luminosity analysis.(DOCX)Click here for additional data file.


Background. Recent intravascular ultrasound (IVUS) studies have demonstrated that hypoechoic plaque with deep ultrasound attenuation despite absence of bright calcium is common in acute coronary syndrome. Such “attenuated plaque” may be an IVUS characteristic of unstable lesion. Methods. We used optical coherence tomography (OCT) in 104 patients with unstable angina to compare lesion characteristics between IVUS-detected attenuated plaque and nonattenuated plaque. Results. IVUS-detected attenuated plaque was observed in 41 (39%) patients. OCT-detected lipidic plaque , thin-cap fibroatheroma , plaque rupture , and intracoronary thrombus  were more often seen in IVUS-detected attenuated plaques compared with nonattenuated plaques. Conclusions. IVUS-detected attenuated plaque has many characteristics of unstable coronary lesion. The presence of attended plaque might be an important marker of lesion instability.  The micron-scale resolution of OCT allows excellent differentiation of atherosclerotic plaque components such as fibrous, fibrocalcific, and lipid [Intravascular ultrasound (IVUS) is widely used to evaluate vulnerable plaques in patients with coronary artery disease. Attenuated plaque, which is defined as a hypoechoic plaque with deep ultrasound attenuation despite absence of bright calcium, is more often observed in patients with acute coronary syndrome than in those with stable angina . Recent nd lipid , 4. Morend lipid , 6. TherA total of 104 patients with primary unstable angina pectoris (UAP) who had de novo coronary lesions and underwent OCT and IVUS to evaluate culprit lesion morphologies in Wakayama Medical University, Wakayama, Japan, and Social Insurance Kinan Hospital, Wakayama, Japan, were studied retrospectively. UAP was defined according to the Braunwald clinical classification , 8: clasμm thick [OCT  was performed before any intervention and IVUS imaging. We used a continuous-flushing (nonocclusive) method for OCT image acquisition. A 0.016-inch wire-type imaging catheter was positioned distal to the culprit lesion. To remove blood cells from the field of view, a mixture of commercially available dextran 40 and lactated Ringer solution was infused from the guiding catheter at 2.5 to 4.5 mL/s with an injector pump. The culprit lesion was imaged with a motorized pull-back device travelling at 1 mm/s. Continuous OCT images were stored digitally for subsequent analysis. The OCT analysis was performed by 2 independent observers who were blinded to the IVUS findings. When there was any discordance between the observers, a consensus reading was obtained. As reported previously , 4, fibrAfter intracoronary administration of nitroglycerin 0.2 mg), the IVUS examination was performed using a commercial scanner  that consisted of a 40-MHz transducer. The IVUS catheter was advanced beyond the culprit lesion followed by automatic transducer pull back (at 0.5 mm/s) to the aorto-ostial junction. IVUS images were recorded onto a DVD for offline analysis. Using custom-built software, IVUS images were analyzed by 2 independent observers who were blinded to the OCT findings. When there was any discordance between the observers, a consensus reading was obtained. IVUS-detected attenuated plaque was defined as a hypoechoic plaque with deep ultrasound attenuation despite absence of bright calcium , 2. Repr mg, the Quantitative coronary angiography analysis was performed using the Cardiovascular Measurement System . Angiographic lesion morphology was classified according to the ACC/AHA classification . The pert-test. All analyses required a P < 0.05 for statistical significance.Statistical analysis was performed using Statview 5.0.1 . Categorical variables were presented as absolute numbers and percentages, with comparison using chi square statistics or Fisher exact test, if there was an expected cell value <5. Continuous variables were presented as mean ± standard deviation and were compared using Student's P = 0.002) and class II  was significantly lower in the attenuated plaque group compared with the nonattenuated plaque group, the frequency of class III  was significantly higher in the attenuated plaque group. Angiographic lesion characteristics were similar between the 2 groups.IVUS-detected attenuated plaque was observed in 41 (39%) patients with UAP. Clinical and angiographic characteristics are listed in 2 versus 2.6 ± 1.0 mm2, P = 0.676) was similar between attenuated plaques and nonattenuated plaques. At the minimum lumen area site, EEM area , P&M area , plaque burden , and frequency of positive remodeling  were significantly greater in attenuated plaques compared with nonattenuated plaques.IVUS findings are summarized in P < 0.001) was significantly higher in attenuated plaques compared with nonattenuated plaques, while the frequency of fibrocalcific plaque  and fibrotic plaque  was significantly lower in attenuated plaques. In the lipidic plaques, fibrous cap thickness , plaque rupture , and intracoronary thrombus  were more often seen in attenuated plaques compared with nonattenuated plaques.OCT findings are summarized in In this IVUS and OCT study, we demonstrated that IVUS-detected attenuated plaques are associated with lipidic plaque, TCFA, plaque rupture, and intracoronary thrombus. Our results suggest the biologic instability of these lesions.P < 0.001) and were associated with a higher C-reactive protein level and more complex lesion morphology on angiography [IVUS is a useful tool for identification of high-risk or unstable lesion morphologies. Previous IVUS data have indicated that culprit lesions in acute coronary syndromes often have a large plaque burden , 14, posiography . In theiiography . Okura eiography , most stκ = 0.83–0.84) as well as excellent sensitivity and specificity: 71–79% and 97–98% for fibrous plaques; 95–96% and 97% for fibrocalcific plaques; 90–94% and 90–92% for lipid-rich plaques, respectively [P = 0.021) and intracoronary thrombus  [r = 0.90; P < 0.001) [OCT has an excellent ability for coronary plaque characterization and vulnerable plaque detection. Histological validation of OCT revealed good intra- and interobserver reliability κ = 0.8–0.84 as Recent studies have shown the mechanisms of ultrasound attenuation in atherosclerotic plaques. In human cadaver coronary arteries, Yamada et al. demonstrated that the percentage of necrotic core area was significantly greater in attenuated plaques than nonattenuated plaques . In lineThere were several limitations. First, this was a retrospective study in nonconsecutive UAP patient. Therefore, the prevalence, clinical feature, and prognostic implication of IVUS-detected attenuated plaque need to be examined by a larger population study. Second, we only included UAP patients. In the examination of attenuated plaque and its potential relationship to plaque vulnerability, our study is limited by the lack of a comparison group with stable presentation. Third, in the IVUS analysis, acoustic shadowing in attenuated plaques may interfere with calculation of remodeling and plaque burden. Fourth, in the OCT analysis, signal attenuation of lipidic tissue or thrombus may preclude visualization and measurement of the entire atherosclerotic plaque. Finally, we used 40-MHz IVUS transducers according to the most previous studies of attenuated plaques. Our results may be not applicable for the IVUS images acquired by other frequency transducers, because the IVUS frequency can affect the degrees of signal penetration .IVUS-detected attenuated plaque has many characteristics of unstable coronary lesion. The presence of attended plaque might be an important marker of lesion instability.


Psathyrella velutina lectin) bound an unique N-acetylglucosamine-terminated N-glycans on “CSF-type” Tf whereas SSA (Sambucus sieboldiana agglutinin) bound α2,6-N-acetylneuraminic acid-terminated N-glycans on “serum-type” Tf. PVL-TfAb ELISA of 0.5 μL CSF samples detected “CSF-type” Tf but not “serum-type” Tf whereas SSA-TfAb ELISA detected “serum-type” Tf but not “CSF-type” Tf, demonstrating the specificity of the lectin-TfAb ELISAs. In idiopathic normal pressure hydrocephalus (iNPH), a senile dementia associated with ventriculomegaly, amounts of the SSA-reactive Tf were significantly higher than in non-iNPH patients, indicating that Tf glycan analysis by the high-throughput lectin-TfAb ELISAs could become practical diagnostic tools for iNPH. The lectin-antibody ELISAs of CSF proteins might be useful for diagnosis of the other neurological diseases.We have established high-throughput lectin-antibody ELISAs to measure different glycans on transferrin (Tf) in cerebrospinal fluid (CSF) using lectins and an anti-transferrin antibody (TfAb). Lectin blot and precipitation analysis of CSF revealed that PVL ( CSF , which circulates within the ventricles of the brain and subarachnoid space, reflects the physiological and pathological conditions of the central nervous system . In factWe previously designated Tf-1 and Tf-2 as two isoforms of transferrin in CSF which are separable by SDS-PAGE and showed that the Tf-2/Tf-1 ratio is higher in iNPH patients than in non-iNPH patients , 4. In tβ1,4-N-acetylglucosamine (GlcNAc) and core α1,6-Fucose [α2,6-N-acetylneuraminic acid (NeuAc) [α2,6-NeuAc on Tf-2, we chose PVL (Psathyrella velutina lectin) and SSA (Sambucus sieboldiana agglutinin) to detect Tf-1 and Tf-2 respectively. Our data showed that the lectin-TfAb ELISAs distinguish the two isoforms to be quantified and that the amounts of SSA-reactive Tf were higher in iNPH patients than in non-iNPH patients. Our newly established lectin-TfAb ELISAs are high throughput methods to measure “glycoforms” of transferrin which might be practical for clinical use. To establish a high throughput method to distinguish Tf-1 and Tf-2, we developed ELISAs using a combination of lectins and an anti-Tf antibody (TfAb). Tf-1 has a “CSF-type” biantennary asialo- and agalacto-complex type N-glycans with bisecting 6-Fucose , 8, wher (NeuAc) , 9. BecaThis study included 28 iNPH patients comprising 14 males and 14 females aged 75.2 ± 6.1 years (mean ± SD) and 18 non-iNPH patients comprising 10 males and 8 females aged 74.9 ± 5.2 years . The iNP, incubated sequentially with an anti-transferrin antibody  and a horseradish peroxidase-labeled anti-goat IgG , and developed using a Super Signal West Dura Extended Duration Substrate . For lectin blotting, the transferred membrane was blocked in 1% BSA, incubated with a biotinylated PVL or biotinylated SSA  followed by a horseradish peroxidase-labeled streptavidin , and developed.CSF samples were dissolved in Laemmli buffer, boiled for 3 min, and loaded onto SDS-polyacrylamide gels . After SDS-PAGE, the proteins were transferred to a nitrocellulose membrane . The membrane was blocked in 3% skim milkCSF was incubated with SSA-agarose (Seikagaku Corporation), and the bound proteins were precipitated by centrifugation. The unbound proteins were further incubated with PVL-agarose (Seikagaku Corporation), and the bound proteins were precipitated.Tf-1 was purified from human CSF as described before . Brieflyμg PVL (Seikagaku corporation) at 4°C overnight and blocked with 0.4% BlockAce . Purified Tf-1 was used as the standard. The standards and CSF samples were appropriately diluted with PBST , applied to the plate, and incubated at 4°C overnight. After three washes with PBST, the plate was incubated sequentially with anti-Tf antibody  and horseradish peroxidase-labeled anti-goat IgG . After three washes with PBST, the wells were incubated with TMB solution , and 1 N HCl was added to stop the reaction. Absorbances at 450 nm were measured by a plate reader (Bio-Rad Laboratories). CV (Coefficient of variation) of PVL-ELISA was 5.36%.For PVL-TfAb ELISA, a 96-well C8 Maxisorp Nunc immuno module plate  was coated with 2.5  For SSA-TfAb ELISA, anti-Tf antibodies  were pretreated with 1.4 mM sodium periodate to abolish SSA epitopes on the antibody and coated on a 96-well plate. Human reference serum (Bethyl laboratories) containing 3 mg/mL serum Tf was used as the standard. The standards and CSF samples were appropriately diluted with TBS  containing 0.05% Tween 20 and 0.5 mM EDTA and applied to the plate and incubated at 4°C overnight. After three washes with TBST, the plate was incubated sequentially with a biotin-SSA (Seikagaku Corporation) and a horseradish peroxidase-labeled streptavidin (Takara). After three washes with TBST, the plate was incubated with the TMB substrate, and the absorbances at 450 nm were measured. CV of SSA-ELISA was 3.65%.t-test and Mann-Whitney U test, respectively.Data were analyzed with SPSS version 17 . Amounts of SSA-Tf and PVL-Tf were analyzed by the Student's α2,6-NeuAc on Tf2 but not sugars on Tf-1. These band signals detected by PVL and SSA . In such cases, the lectin-antibody ELISAs are more useful to detect specific glycoforms than immunoblotting. Based on the concept that searches for biomarkers should involve not only “proteomics” but also “glycoproteomics” , 12, we We have developed the PVL-TfAb ELISA and SSA-TfAb ELISA to measure Tf-1 and Tf-2, respectively. Amounts of the SSA-reactive Tf were significantly higher in CSF of iNPH patients than in non-iNPH patients, suggesting that the lectin-TfAb ELISAs are promising high throughput methods for diagnosing iNPH. The lectin-antibody ELISAs might be useful for a CSF biomarker study of neurological diseases.


In round microchannels hematocrit was measured in parent and daughter branch at baseline and after addition of DRP. At DRP concentrations of 5 and 10 ppm, the plasma skimming effect in the daughter branch was eliminated, as parent and daughter branch hematocrit were equivalent, compared to a significantly lowered hematocrit in the daughter branch without DRPs. In anesthetized rats (N=11) CFL was measured in the cremaster muscle tissue in arterioles with a diameter of 32.6 ± 1.7 µm. In the control group  there was a significant increase in CFL in time compared to corresponding baseline. Addition of DRP at 1 ppm (N=5) reduced CFL significantly compared to corresponding baseline and the control group. After DRP administration the CFL reduced to about 85% of baseline at 5, 15, 25 and 35 minutes after DRP infusion was complete. These in vivo and in vitro findings demonstrate that DRPs induce a reduction in CFL width and plasma skimming in the microvasculature. This may lead to an increase of RBC flux into the capillary bed, and thus explain previous observations of a DRP mediated enhancement of capillary perfusion.Drag-reducing polymers (DRPs) significantly increase blood flow, tissue perfusion, and tissue oxygenation in various animal models. In rectangular channel microfluidic systems, DRPs were found to significantly reduce the near-wall cell-free layer (CFL) as well as modify traffic of red blood cells (RBC) into microchannel branches. In the current study we further investigated the mechanism by which DRP enhances microvascular perfusion. We studied the effect of various concentrations of DRP on RBC distribution in more relevant round microchannels and the effect of DRP on CFL in the rat cremaster muscle  In a canine model of severe coronary artery stenosis, we have shown that nanomolar concentrations of blood-soluble drag-reducing polymers (DRPs) restored myocardial perfusion to normal, measured using radiolabeled microspheres and myocardial contrast echocardiography (MCE) . This obin vitro observations in a model of a vessel bifurcation, an alternate mechanism was suggested. That is, DRPs diminish flow separations and recirculation zones at vessel bifurcations [in vivo (microvessels) and in vitro (capillary tubes). To shed light on the microvascular mechanisms of DRP, Marhefka et al. [in vitro by observing the traffic of RBC in microfluidic systems. During the flow of RBC suspensions in straight rectangular microchannels with bifurcations and expansions, they observed a redistribution of RBCs and a reduction in both plasma layer width and plasma skimming  aftein vitro measurements in round bifurcating microchannels using several concentrations of DRP. Our goal was to determine whether the observed redistribution of RBCs occurs in a more physiologically relevant setup using round channels, instead of rectangular channels as reported previously [in vitro occurs in vivo. To determine the effect of DRP in vivo the plasma layer, or cell-free layer (CFL), was measured in microvessels of the rat cremaster tissue before and after the infusion of DRP. We found, using round microchannels, a reduction in plasma skimming with DRP. In addition, we observed that DRP causes a reduction in CFL in an in vivo animal model of the rat cremaster muscle.In the current study, we performed eviously . FurtherDRP solution was comprised of polyethylene oxide , with an average molecular weight (MW) of 4000 kDa. PEO was dissolved in saline at a concentration of 0.1% and then dialyzed against normal saline for 24 hours using a membrane with 50 kDa MW cutoff . Viscosity of the PEO solution was measured by a rotational viscometer  to confirm complete dissolution and rule out mechanical degradation of the polymer during preparation. The stock PEO solution of 1000 ppm concentration was diluted to a concentration of 50 ppm with saline and mixed for 1–2 hours on a slow rocker prior to use.Whole bovine blood was obtained from a local slaughterhouse  with their permission to be used for research. Heparin  was added to maintain anticoagulation. Blood was washed as described by Johnson et al. . In shorExperiments were carried out using round microchannels with a diameter of 50 µm constructed using a suspended-wire technique  see Fig. The micThe flow pump was started, filling the feed loop with the RBC suspension, after which the withdrawal pumps were started. The system was allowed to run initially until both the polyethylene tubing between the microchannel and capillary tube, and the capillary tube itself, were filled. After the capillary tubes in each branch were full, the withdrawal pumps were temporarily stopped, the capillary tubes removed, and one end was sealed using Critoseal®Capillary Tube Sealant . After the initial stop, only the capillary tubes were allowed to fill prior to stopping. The tubing connected to the microchannel had continuous flow, as the feed loop was continued throughout to prevent thrombus formation. Capillary tubes from the parent branch were separated from those taken from the daughter branch, and all were centrifuged. After centrifugation, hematocrit was calculated by dividing the height of RBC column by the total height of the contents of the capillary tube and then multiplying by 100 to obtain percentage. These steps were repeated for RBC suspensions without DRP initially (N=91), followed by those with DRP at concentrations of 10 ppm (N=17), 5 ppm (N = 85), and 1 ppm (N=29). All hematocrit values were recorded and analyzed. All experiments were conducted at room temperature.All experimental protocols were approved by the Institutional Animal Care and Use Committee of University of Pittsburgh. Young Wistar male rats (N = 11) weighing 147 ± 14 g were anesthetized with isoflurane (3%) and maintained at 1–2% isoflurane. A 24G catheter was placed in the carotid artery and connected to a fluid filled catheter. Systemic blood pressure was recorded continuously using a Ponemah data acquisition system . The right external jugular vein was cannulated with a 24G angiocatheter for DRP or saline infusion. An abdominal midline incision was made, the distal abdominal aorta was isolated and encircled with a 1 mm flow probe , and the incision was closed. The cremaster muscle was prepared for intravital microscopy as previously described . BrieflyFollowing surgery, the completed preparation was transferred to the stage of an intravital inverted microscope  linked to an ORCA-ER CCD camera  recording at 8.3 frames per second. Recordings were made using the software package HCImage . Microvessels were observed using bright-field  microscopy : 0.8) for the examination of CFL. A blue filter  was used to enhance the contrast between the RBCs and the background field. Cremaster muscle arterioles were examined with a 60× objective lens .After being placed on the microscope stage, the preparation was allowed to equilibrate for 30 min, during which time the arteriolar network was scanned to find suitable arterioles with a diameter of 30 µm. In each animal one vessel was selected and recordings were made at baseline and 5 minutes after completion of DRP infusion and repeated every 10 minutes for 30 minutes. DRP solution (N = 5) was infused over 6 minutes (about 0.15–0.3 ml) to a final blood concentration of 1 ppm, similar to what was used in our previous study . In an aAll digital image processing procedures were performed with a commercially available image processing software package . For each recording, 4 frames were selected. To correct for movement of the tissue, the images were aligned using subpixel image registration by cross-correlation . The 4 iin vitro setup was determined using an unpaired t-test. In vivo, effects of DRP on CFL, blood pressure, flow and vessel diameter within a group were analyzed using a Kruskal-Wallis test followed by Dunn’s multiple comparison post-hoc test. The effect of DRP on CFL comparing control with treated animals was assessed using an unpaired t-test. Results were considered statistically significant with P<0.05. Summary data are reported as means ± SEM, unless otherwise indicated.Significance of the effect of DRP on hematocrit in the In the control experiment the hematocrit measured in the daughter branch was 89.7 ± 1.0% of the hematocrit in the parent branch (P<0.001). After administration of 1 ppm of DRP there was a trend towards an increase in hematocrit in the daughter branch (P=0.08) compared to the control experiment see , but theBaseline measurements of carotid pressure and aortic flow were 90.1 ± 3.9 mmHg and 11.3 ± 1.0 ml/min. Following DRP infusion, there was no statistically significant difference in these parameters at any of the time points up to 35 minutes after DRP infusion. Similarly these values were unchanged after saline infusion. Hematocrit taken during surgery  and after completion of the experiment  was not statistically different comparing the control group with the animals treated with DRP. Vessel diameter was 32.6 ± 1.7 µm (range 25–44 µm) at baseline and did not change during the experiment for the animals treated with DRP or saline.An example of a frame before and after analysis of the intensity profiles is given in The CFL data was normalized to baseline. Comparing the normalized CFL data, a significant difference was found comparing the control animals to those treated with DRP (P<0.001) for each time point after baseline. The CFL observed in the control animals was significantly larger compared to those animals treated with DRP. When comparing baseline to the different time points, the CFL was significantly reduced in the animals treated with DRP. In the control animals the CFL was significantly increased compared to baseline at 15, 25 and 35 minutes after the infusion of saline (P<0.05), except at the 5 minute time-point .in vitro observations showing a decrease in CFL with DRP do occur in vivo. Based on our in vitro and in vivo observations, this suggests that DRP reduces plasma skimming, increases arteriolar side branch hematocrit, thereby diverting more RBCs into the capillary bed. This redistribution of RBCs due to a reduction in plasma layer width within the microvascular network [In this study we have shown that DRP eliminates plasma skimming in a 50 µm T-bifurcation model, consisting of cylindrical microchannels. This effect aligns with previous observations showing a reduction in CFL width in square microchannels using flow visualization techniques ,12. The  network  may explin vitro model consisting of rectangular channels, however, the vasculature consists of round-shaped vessels. When implementing the experiment in a model with round microchannels we observed a similar phenomenon, a redistribution of RBCs increasing the hematocrit in a side branch up to the level of the parent vessel. Importantly, this effect was DRP concentration dependent, at a concentration of 1 ppm there was a trend towards an increase in hematocrit in the side branch and at 5 and 10 ppm there was no longer a difference between hematocrit in the main channel and a side branch.Infusion of DRP has been associated with increased survival after hemorrhagic shock ,6,8, impin vivo microcirculatory hematocrit are found to be significantly smaller than the systemic hematocrit [in vitro experiments was equal to the hematocrit found in arterioles with a comparable diameter (50 µm) in the rat cremaster [in vitro studies using rectangular microchannels [In these studies a hematocrit of 30% was chosen since matocrit -31 due tremaster . Similarchannels ,33 we usin vitro microchannel models. However, microvessels are distinct from microchannels in that they are elastic, lined with endothelium, relatively short, and may be irregular in shape. Furthermore, CFL formation in arteriolar bifurcations has different characteristics compared to CFL formation in microchannels. Ong and coworkers observed spatial variability of the CFL formation in the vicinity of an arteriolar bifurcation due to blood flow separation at the bifurcation, leading to heterogeneities of the layer formation not only within individual vessel segments but also between downstream vessels at the bifurcation [in vitro setup, therefore, cannot be completely extrapolated to an in vivo setting. To further elucidate potential mechanisms behind the DRP effects on the microcirculation we performed experiments in the rat cremaster microvessels in vivo where one group was treated with saline and the other group with DRP (1 ppm). Importantly, the CFL was measured at a distance exceeding two vessel diameters away from any bifurcation, to minimize any potential confounding effects of the bifurcation on the CFL [So far, the effect of DRP on perfusion has been studied using urcation ,35. Data the CFL .We used the same concentration of DRP that successfully enhanced perfusion in the canine myocardium seen in our previous study ,2. Addinin vitro and in vivo observations, we hypothesize that a reduced CFL results in less plasma skimming and a more homogeneous flow and hematocrit distribution [The width of the CFL is influenced by many parameters, such as hematocrit, presence of endothelial glycocalyx, vessel diameter, RBC aggregation and deformability, and mean flow velocity . In our ribution  throughoribution . The entribution ,40. DRP ribution ,42. Furtribution , which iribution -46. The ribution ,47. Neveribution . TherefoAnalysis of CFL was based on the technique developed by Kim et al. ,20. In tin vitro setup, a significant effect of DRP on CFL was observed at a concentration of 1 ppm in the rat cremaster tissue. Since it was proven that the change in CFL occurs as a result of DRP administration, and the effect was obtained at 1 ppm, it was decided to not repeat the experiments at higher DRP concentrations. However, studies to demonstrate a dose response effect in vivo would be of value. Based on preliminary data on the effect of DRP concentration (1-5 ppm) on RBC velocity, aortic blood pressure and blood flow, a further change in CFL with increasing concentration of DRP might be expected. Furthermore, in the in vitro studies we found a dose dependent effect of DRP on the magnitude of plasma skimming, which suggests that there is a dose dependent effect on the CFL.Unlike the We used a camera with a frame rate of 8.3 frames per second. Mean centerline RBC velocity was between 4-5.5 mm/sec and although velocity of the RBCs was less near the vessel wall, smearing of the RBCs has been observed. Consequently, some RBCs look elongated since they moved during recording of the frame. However, the low frame rate worked as a low pass filter, resulting in a better averaged CFL width estimation due to a temporal averaging within a single frame. The spatial resolution of the images was 9.30 pixels per micrometer. The minimum difference in CFL that could be detected is, therefore, 0.11µm. The change in CFL observed in the current study was 0.5µm and in different direction for control (increase in CFL) as for DRP treated animals (reduction in CFL). Thus, the spatial resolution formed no limitation for detecting changes in CFL width.When analyzing the data, a part of the vessel was selected that was relatively straight. Small irregularities in the shape of the vessel were not corrected for. Therefore, it is possible that not all measurements of the distance between the inner vessel wall and the RBC column were exactly perpendicular to the vessel wall. However, when comparing our measurements we did use the same part of the vessel for all 5 stages in our experiment, therefore any error introduced by irregularities in vessel wall will have no effect on the outcome of our study. The absolute CFL dimension might have been influenced by not taking irregularities in vessel wall into account but, as mentioned in the discussion, our data is well within the range reported by others ,20.Last, the hematocrit values measured in our study in both the control group and DRP treated animals are lower as those values reported in literature for rats (35-45% hematocrit ). Duringin vivo animal model of the rat cremaster muscle by nanomolar concentrations of intravenous DRPs. Our in vitro and in vivo findings taken together, suggest that through a reduction in plasma skimming, DRPs cause redistribution of RBCs within the microvascular network, which would promote delivery of RBCs to the capillary bed, and may explain the previously observed DRP induced increase in capillary perfusion. These findings provide novel insight into the microvascular mechanisms of DRPs.The findings of the current study demonstrated 1) reduction in plasma skimming by DRPs using round microchannels, and for the first time, 2) reduction in CFL in an