CaMKIV-/-) were tested in a battery of stress and anxiety-related behavioral tests, to determine if CaMKIV plays a role in emotional behavior. CaMKIV-/-exhibited a decrease in anxiety-like behavior in both the elevated plus maze and dark-light emergence tests when compared to wild-type mice. Both the acoustic startle response and prepulse inhibition of startle were decreased with the deletion of CaMKIV. In addition, CaMKIV-/- mice displayed a lack of stress-induced analgesia following restraint or cold swim stress. Our results demonstrate a key role for CaMKIV in anxiety and stress-related behavior.Calcium-calmodulin-dependent protein kinase IV (CaMKIV) phosphorylates the major transcription factor cyclic AMP-response element binding protein (CREB), which plays a role in emotional behavior. Here, CaMKIV knockout mice ( Among different Ca2+-dependent protein kinases, CaMKIV is detected predominantly in the nuclei of neurons [Calcium-calmodulin-dependent protein kinase IV (CaMKIV) plays a role in the activity-dependent phosphorylation of cyclic AMP-responsive element binding protein (CREB) and CRE modulator (CREM), which regulate the expression of genes involved in neuroplasticity , learnin neurons ,11, ther neurons .CaMKIV is normally expressed in the amygdala and hippocampus, two brain structures involved in the regulation of anxiety and CaMKIV deficient mice exhibit defects in contextual and auditory fear memory . A recenCaMKIV-/- mice exhibit decreased anxiety-like behaviors in several anxiety paradigms and develop less stress-induced analgesia. Our results demonstrate a key role for CaMKIV in mediating changes in anxiety and stress-related behaviors.Previous studies have implicated that both CREB  and CREMCaMKIV-/- mice were derived as described [ad libitum. All experiments were carried out in accordance with the rules and regulations of the Animal Care and Use Committee at the University of Toronto. All efforts were made to minimize the animal's suffering and to reduce the number of animals used. No visual difference between C57BL/6 and CaMKIV-/- mice was noticeable and experiments were performed blind when possible.All subjects were 8–12 weeks old male mice. The escribed  and bredThe elevated plus maze  consists of two open arms and two closed arms situated opposite each other and separated by a 6 cm square center platform. Each runway is 6 cm wide and 35 cm long. The open arms have lips that are 0.5 cm high and the closed arms are surrounded on three sides by 20 cm walls. The floors and walls are black polypropylene. For each test, the animal was placed in the center square and allowed to move freely for five minutes. Open arm entries were defined as the mouse having all four paws onto the open arm. The number of entries and time spent in each arm was recorded.The testing apparatus consisted of a rectangular Plexiglas box (44 × 8.5 × 25 cm) equally divided into a light, open topped, compartment connected by a door (17 cm in height) to a dark, closed topped, opaque compartment. Each mouse was placed in the light box and was allowed 10 sec to explore before the door to the dark box was opened. Each animal was tested for 10 min. The time spent in the light compartment and the number of light/dark transitions were recorded.The apparatus consisted of a black opaque plastic box (13 × 11 × 8.5 cm) with a small opening (3 × 6 cm) placed along one side of the open field evenly illuminated by white overhead lighting. The exit was faced out into the open field. Mice were individually placed into the box with the exit blocked for a 5 min habituation period. At the end of the habituation period, the exit was opened and the mice were allowed to freely explore the open field for 5 min. The latency to emerge from the box, time out of the box, time spent assessing the open field (scanning the open field with the head but less than four paws out of the box) and box/open field transitions were recorded.Testing was conducted in a startle chamber from Med Associates . The startle chamber consists of a nonrestrictive Plexiglas cylinder (4.4 cm) in diameter, resting on a platform in a ventilated and sound attenuating chamber. A high-frequency speaker mounted behind the cylinder produced all the acoustic and prepulse stimuli. Mouse movements were detected and transduced by a stablimeter mounted under the cylinder and digitized and stored by a computer and interface assembly. Startling stimulus began at 80 dB and 1 ms readings were recorded to obtain the amplitude of the animal's startle or prepulse response to each stimulus. For acoustic startle, mice were acclimated in the chamber for 20 min and 80 trials of 80, 85, 90, 95, 100, 110 and 120 dB noise bursts were presented over a 45-min test session. The inter-trial interval varied randomly from 10 to 20 s, with an average of 15 s. For PPI, each test session consisted of 55 trials and was initiated with a 10-min acclimation period. Testing began after the initial exploratory behavior had diminished. Three different trial types were presented at random: 20 ms prepulse of 80, 90 or 100 dB, 100 ms before a startle noise burst (112 dB), startle noise burst (112 dB) alone and background noise alone. For both acoustic startle and PPI the background noise was at 70 dB.versus time) was used.To induce stress, mice were forced to swim in water (10°C) for 3 min or were restrained for 30 min. Animals were individually restrained in small cylindrical tubes with a diameter slightly larger than a mouse's body. Responses to the hot-plate and tail-flick were measured at different time points up to 60 min after stress. Data are presented as the mean response latency (s) or maximum possible inhibition (MPI = (response latency – baseline response latency)/ (cut-off time – baseline response latency) × 100). The total effect of stress over time, the area under the curve  and purified with RNeasy mini kit (Qiagen). RNA quality was checked visually on an agarose gel and also by Agilent BioAnalyzer. Combined RNA was subjected to gene chip analysis (Affymetrix Mouse 430 2.0). First-strand synthesis, fragmentation, hybridization and washing were done according to GeneChip (Affymetrix Inc.) protocol. The array was scanned, and information was extracted using the GeneChip Expression Analysis program (Affymetrix) and analyzed using the GeneChip Operating System .Total RNA was isolated from the forebrains of two C57BL/6 or CaMKIV-/- mice previously extracted for the GeneChip experiment (n = 2 for C57BL/6 and CaMKIV-/- mice) using SuperScript III RT (Invitrogen) with oligo(dT) as a primer. The primers for RT-PCR were as follows: Oxytocin: sense, 5'-TTGCTGCCTGCTTGGCTTAC-3', antisense, 5'-TATTCCCAGAAAGTGGGCTC-3', arginine vasopressin: sense, 5'- ACACTACGCTCTCCGCTTGT-3', antisense, 5'- GGGCAGGTAGTTCTCCTCCT-3', Transthyretin: sense, 5'- ATGGTCAAAGTCCTGGATGC-3', antisense, 5'- CAGAGTCGTTGGCTGTGAAA-3', connexin 43: sense, 5'- GGACTGCTTCCTCTCACGTC-3', antisense, 5'- CAGCTTGTACCCAGGAGGAG-3' and synaptotagmin 1: sense 5'-CAATACTGCCATTCCCTCGT-3', antisense, 5'- GTAGCAGGCTCACCTTCCTG-3'. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was ampli?ed as an internal control by using the primer sets: sense, 5-AACGACCCCTTCATTGAC-3' antisense 5'-TCCACGACATACTCAGCAC-3'. PCR conditions were adjusted to be in a linear range of amplification.First strand-cDNAs were synthesized from total RNA isolated from the forebrains of C57BL/6 and P < 0.05 was considered statistically significant.Results were analyzed by t-test, paired t-test, ONE-WAY ANOVA, TWO-WAY ANOVA followed by post-hoc Student-Newman-Keuls test to identify significant differences. All data are expressed as mean ± S.E.M. In all cases, CaMKIV-/- mice were tested in the elevated plus-maze, dark-light emergence test, light/dark box, acoustic startle and for the prepulse inhibition of startle. Previous studies showed that general locomotor activity was similar between CaMKIV-/- mice in an open field when compared to wild-type mice, however, CaMKIV-/- mice spent significantly more time exploring the center of the open field, which is indicative of a reduction in anxiety-like behavior [CaMKIV-/- mice, we used the elevated plus maze, which is a well-validated, conventional test for anxiety-related behavior in mice [CaMKIV-/- mice spent significantly more time in the open arms of the plus-maze  and displayed an increase in the number of open arm visits  when compared to wild-type mice. The number of entries into the closed arms  and the total number of arm entries was also significantly increased , suggesting an increase in locomotor activity.To determine if CaMKIV plays a role in the expression of anxiety-like behaviors, behavior . To furt in mice . In thisCaMKIV-/- mice in the dark-light emergence test [CaMKIV-/- mice again displayed a significant decrease in anxiety-like behavior. We found that the overall time spent in the open field was significantly greater in CaMKIV-/- mice . We further examined changes in anxiety-related behavior using the light/dark box [CaMKIV-/- mice . Since this test is based on a mouse's natural aversion to brightly lit spaces, an overall decrease in exploration by CaMKIV-/- mice may represent a reduction in conflict between the two chambers. Although CaMKIV-/- mice did not display a consistent anxiety-like phenotype in the light/dark box paradigm, evidence suggests that similar rodent behavioral tests may measure different forms of anxiety-like behavior [CaMKIV-/- mice.To strengthen our hypothesis that CaMKIV may play a role in the regulation of anxiety-like behaviors, we tested the performance of nce test . CaMKIV-dark box . Anxiogedark box . The totice Fig. . Howeverbehavior ,22. Prevbehavior . TherefoCaMKIV-/- mice displayed a decrease in baseline acoustic startle amplitudes . Similarly, CaMKIV-/- mice showed a significant reduction in prepulse inhibition of the startle reflex at prepulse intensities of 90 and 100 dB . These results further support our hypothesis that CaMKIV may play a role in emotional behaviors.The acoustic startle response is elicited by a sudden loud acoustic stimulus and is characterized by a coordinated contraction of the muscles of the neck and extremities. The percent prepulse inhibition (PPI) is an index of sensorimotor gating. We examined the acoustic startle response and PPI in mice lacking CaMKIV. Consistent with a reduction in anxiety-like behaviors, CaMKIV-/- and wild-type mice were tested for stress-induced analgesia after either restraint or swim stress. Consistent with previous studies [CaMKIV-/- mice failed to show any analgesia in both tests (P < 0.01 for both tests and area under the curve P < 0.001 and P < 0.05 for hotplate and tail-flick tests respectively). We can exclude the possibility that this is due to altered spinal nociceptive transmission since CaMKIV-/- mice have unaltered baseline tail-flick and hotplate responses [P < 0.05 comparisons of CaMKIV-/- and wild-type mice using area under the curve). Here, we measured behavioral nociceptive responses on the hot-plate after animals were forced to swim in cold water. CaMKIV-/- mice exhibited significantly reduced stress-induced analgesia when compared to wild-type controls. Taken together, these results suggest a role for CaMKIV in the regulation of stress-induced analgesia.To determine if CaMKIV plays a role in stress-induced behavioral changes,  studies , restraiesponses . SimilarCaMKIV-/- mice to uncover any changes in the expression levels of genes related to emotional behavior. We compared gene expression profiles between CaMKIV-/- and wild-type mice and found 233 genes to be differentially expressed with lower expression levels in CaMKIV-/- mice than in wild-type animals. We filtered out the top five anxiety/stress-related genes among down-regulated genes based on previous literature . To confirm this GeneChip data, reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure the mRNA expression level of oxytocin in CaMKIV-/- mice (Fig. CaMKIV-/-. This suggests that, as an upstream regulator of CREB, CaMKIV may play a role in the regulation of anxiety-related genes such as oxytocin. Since oxytocin has been shown to mediate stress-induced analgesia in mice [CaMKIV-/- mice.Several studies show that altering CREB function can change anxiety-like behaviors ,25 and sre Table . Of part in mice  and our CaMKIV-/- mice display reduced anxiety-like behaviors in the elevated plus-maze, dark-light emergence test, and in the acoustic startle reflex and PPI. Furthermore, these mice lack stress-induced analgesia induced by forced swim or restraint stress.In the present study, we show that the genetic disruption of CaMKIV in mice results in a decrease in anxiety-like behavior and the abolishment of stress-induced analgesia. 2+-calmodulin-dependent protein kinase pathway. The nuclear location of CaMKIV, its ability to phosphorylate CREB and its broad expression throughout forebrains areas suggests that CaMKIV may play an important role in higher brain function. CaMKIV may also play a role in CREB phosphorylation by modulating other kinase pathways. Studies in cell culture systems demonstrated the regulatory role of CaMKIV in MAP kinase and cAMP pathways [Several kinase cascades regulate CREB function in the CNS ,9, thesepathways ,29. Distpathways . In-vitrpathways .CaMKIV-/- mice displayed reduced anxiety-like behavior by spending more time in the open arms of the elevated plus-maze, which correlates with the phenotype of the CREM mutant mice. However, mice lacking CaMKIV did not show hyperlocomotor activities in the open field [CaMKIV-/- mice made significantly more entries into the open and closed arms of the elevated plus-maze, suggesting an increase in locomotor activity. One explanation for this discrepancy may be because CaMKIV-/- mice have less anxiety so they may spend more time exploring the anxious environment of the elevated plus maze when compared to wild-type mice. These results suggest that CaMKIV-/- mice have a decrease in anxiety in the plus-maze compared to wild-type mice, and hint that CaMKIV may play a role in regulating levels of anxiety. Therefore, the contrasting phenotypes of between CaMKIV, CREB and CREM knockout mice emphasize the complexity of the transcription factor and genetics underlying such emotional behavior. These studies further suggest that CaMKIV may play an important role in anxiety-like behavior through its regulation of CREB since CREB mutant mice displayed alterations in emotional behavior [Previous studies of CREB mutant mice reported an increase in anxiety-like behavior in several behavioral paradigms including the elevated plus-maze, black and white box and open field . Althougen field . Our resbehavior ,7,25.CaMKIV-/- and wild-type mice. We found at least 200 genes down-regulated, five of which were stress/anxiety-related genes (Table CaMKIV-/- mice include the intracellular transportor gene, transthyretin; secretory vesicle protein, synaptotagmin 1, and a gap junction protein, connexin 43. The down-regulation of these genes suggests that the deletion of CaMKIV may affect the expression of genes involved in the regulation of presynaptic terminals and its structure. These genes are shown to be anxiety/stress related and may have other roles in addition to anxiety. The GeneChip data included in this study may help us to find a mechanism for the role of CaMKIV in anxiety and such a mechanism may include oxytocin.Microarray analysis provides us the opportunity to screen for changes in thousands of genes at the same time  and thises Table . Of thesCaMKIV-/- mice, with reduced oxytocin levels, display a lack of stress-induced analgesia. Contrary to observations in oxytocin deficient mice [CaMKIV-/- mice display lower anxiety-like behaviors. Our findings imply that changes in oxytocin expression may contribute to the changes in anxiety level of CaMKIV-/- mice. Thus, it appears that CaMKIV may regulate the expression of many anxiety-related genes, including oxytocin, but that this regulation cannot solely account for the behavioral phenotype. While future studies are needed to thoroughly characterize changes in gene expression that result from the deletion of CaMKIV and how these changes affect behavioral phenotypes, our initial GeneChip data provides a good starting point for the dissection of molecular changes responsible for the anxiety phenotype of CaMKIV-/- mice.Of interest, accumulating evidence suggests that oxytocin possesses anxiolytic properties and is important for stress-induced analgesia -39. Oxytent mice -39, whicOur results suggest that a nuclear protein kinase, with broad forebrain distribution and the ability to affect gene expression through the activation of CREB, plays a role in anxiety-like behavior and that modulation of CaMKIV activity may prove useful in modifying anxious behavior. Future studies are needed to elucidate the role of CaMKIV in the molecular changes involved in the modulation of anxiety-like behaviors.


Elevated BC also predicted increased risk of ST-segment depression ≥0.5 mm among those with at least one episode of that level of ST-segment depression. Carbon monoxide was not a confounder of this association. ST-segment depression, possibly representing myocardial ischemia or inflammation, is associated with increased exposure to particles whose predominant source is traffic.Increased levels of daily ambient particle pollution have been associated with increased risk of cardiovascular morbidity. Black carbon (BC) is a measure of the traffic-related component of particles. We investigated associations between ambient pollution and ST-segment levels in a repeated-measures study including 269 observations on 24 active Boston residents 61–88 years of age, each observed up to 12 times from June through September 1999. The protocol involved continuous Holter electrocardiogram monitoring including 5 min of rest, 5 min of standing, 5 min of exercise outdoors, 5 min of recovery, and 20 cycles of paced breathing. Pollution-associated ST-depression was estimated for a 10th- to 90th-percentile change in BC. We calculated the average ST-segment level, referenced to the P-R isoelectric values, for each portion of the protocol. The mean BC level in the previous 12 hr, and the BC level 5 hr before testing, predicted ST-segment depression in most portions of the protocol, but the effect was strongest in the postexercise periods. During post-exercise rest, an elevated BC level was associated with −0.1 mm ST-segment depression ( A baseline screening questionnaire was administered regarding medications, pulmonary and cardiac symptoms, and smoking history. A resting 12-lead electrocardiogram (ECG) was performed. Exclusion criteria included unstable angina, atrial flutter, atrial fibrillation, or paced rhythm. Each subject was assigned a day of the week and a time of day for weekly testing, with the goal of 12 weekly visits during the summer of 1999. Each week, participants were administered a brief questionnaire regarding chest pain, medication changes, and whether medications had been taken that morning. Continuous Holter monitoring with electrodes in a modified V5 and aVF position was performed using the Marquette Seer Digital Recorder . The protocol  consisteThe digital Holter recordings were downloaded to a MARS Ultra 60 playback system (Marquette Inc.) for analysis. ST-segments were evaluated for the average value for each portion of the protocol and for possible ischemia, defined as reversible horizontal or downsloping ST-segment depression ≥0.5 mm, a level associated with adverse cardiac risk in patients with acute coronary syndrome . Recordi2.5, BC, CO) were collected at a central site within 0.5 km of the residences of the subjects, which were on the same busy street trafficked by diesel-powered buses and trucks as well as cars of commuters. Measurements of sulfur dioxide, ozone, and nitrogen dioxide were obtained from state monitoring sites in Boston. Continuous PM2.5 was measured using a tapered element oscillating microbalance . The TEOM sample filter is heated to 50°C, leading to season-specific temperature-related loss of semivolatile mass. Season-specific calibration factors were used to correct for the losses of mass  low-volume Teflon filter gravimetric measurements.Air pollution measurements . BC data from this instrument, using the internal empirically determined conversion factor, have correlated well with elemental carbon . Analyses were repeated including all study participants to assess the sensitivity of results to the exclusion criteria and to the presence of outliers.The Exposure and Risk Assessment for Fine and Ultrafine Particles in Ambient Air (ULTRA) study has demonstrated the importance of selecting a vulnerable population when seeking to investigate whether pollution influences ECG changes consistent with ischemia . AlthougIn addition to analyses evaluating ST-segment level as a continuous outcome, we analyzed the binary response “ST-segment depression ≥0.5 mm,” defined as a mean ST-segment level for a given portion of the protocol of at least −0.5 mm . This definition differed from that of classic ischemia in that it did not require within-test or within-portion of the protocol reversibility. For this secondary analysis, we fit a logistic regression model with random intercepts to data from those subjects having at least one response of each type (depressed and nondepressed ST-segment) during that particular protocol (13 of 28 study participants contributed data to at least one portion of the protocol).Twenty-four study participants with 269 observations were included in analyses either with continuous or with binary (dichotomous) ST-segment outcomes. We had sufficient observations to evaluate the effects of between-test increases in pollution levels on between-test depression in the mean ST-level for each portion of the protocol. However, we were unable to assess the effect of between-test changes in pollution on the risk of within-test reversible ST-segment depression that fit criteria for ischemia because of the rarity and lack of variability of such events. During the study, only 5 of 28 study participants had ischemic ECG events .Each regression model included an indicator variable for each subject, pollutant concentration, a cubic effect of the mean of the current hour temperature, and a linear trend of time. Other confounders considered included day of week and time of day, which were both highly correlated with the subject indicator variables and were thus dropped from the model. Separate models were fit using lags of 1–24 hr, as well as previous 12 and 24 hr moving averages, of pollution concentration. Finally, models containing multiple pollutant concentration as predictors were fit to account for confounding due to moderate to high correlations among different pollutant concentrations. Multiple lags and moving averages were evaluated to select the best lag structure for temperature and each individual pollutant, and models reflect these evaluations. All statistical analyses were performed using the SAS statistical software package . The conditional linear mixed models were fit using PROC MIXED, whereas the logistic mixed models were fit using PROC NLMIXED (SAS Institute Inc.).Estimates of the effects of BC were scaled to the difference between the 10th and the 90th percentile in levels for the appropriate lag or mean value of BC.The median age of the population was 73, and many participants had cardiac risk factors  or coronary artery disease . As expep = 0.03; 5-hr BC: estimated change = −0.10 mm; p = 0.004), suggesting a pollution effect sustained throughout the protocol. Although they were also consistently negative, associations of ST-segment depression with the mean of BC during the 24-hr before testing were weaker, and the BC levels 2 days before testing had no association with ST-segment depression. There was no effect of air pollution on changes in ST-segment level from the rest to exercise or from the exercise to recovery portions of the protocol. The effects of BC on ST-segment depression were not modified by medication use, diagnosis of coronary artery disease, hypertension, sex, or ethnicity.Individual hourly lag models showed consistent negative associations of ST-segment level with increased BC for the first 12 hr before testing , but witFor the smaller group who had at least 0.5 mm depression at one or more visits, increases in BC were associated with an elevated risk of ST-segment depression ≥0.5 mm, although confidence in the estimates was limited by the smaller numbers of observations . The larIn elderly subjects, we found that increases in levels of ambient BC in the 12 hr before testing were associated with between-week depression in the mean ST-segment levels that was present throughout the testing session, with the strongest effects occurring in the postexercise recovery portions of the protocol, a period of cardiac vulnerability in patients with coronary artery disease . There wAlthough we found pollution to be associated with ST-segment depression sustained throughout the testing session, the Finnish portion of the ULTRA study found associations of pollution with reversible exercise-induced ST-segment depression . The etiAlthough a minority of our subjects had documented coronary disease, many had risk factors predisposing them to subclinical disease and possible ischemia. Particle pollution may decrease myocardial oxygen supply and increase the risk of cardiac ischemia due to epicardial coronary disease through potentially interrelated mechanisms, including systemic inflammation, oxidative stress, endothelial dysfunction, and/or autonomic dysfunction . CoronarRather than causing subclinical ischemia, pollution-associated systemic inflammation may lead to low-grade myocardial inflammation, with associated subtle repolarization changes, including sustained ST-segment depression. A series of epidemiologic studies have found associations of particle pollution with elevation of measures of systemic inflammation, including plasma viscosity , fibrinoIn this same study, in the entire cohort, we found that BC was associated with a decrease in heart rate variability, suggesting traffic-particle–associated autonomic dysfunction . Future work will focus on whether ambient pollution leads to ST-segment depression and autonomic dysregulation through related pathways  or through separate pathways.2.5 and CO in the Finnish study of subjects with stable coronary heart disease who performed repeated biweekly submaximal exercise tests over a 6-month period .BC can be viewed as a surrogate for traffic-related particle pollution; exhaust emissions from diesel-powered vehicles have been identified as the main source of BC or elemental carbon in urban areas . Laden eh period . In that2.5 of ambient origin ].This study was limited by lack of personal exposure measurements for CO and particles. However, ambient levels were measured on the same busy city street as the participant residences, < 0.5 km away, and studies in Boston have shown that ambient concentrations are good surrogates of personal exposures to PMt origin . Moreovet origin . Our abit origin . Our priIn conclusion, in a population of elders susceptible to cardiovascular pollution effects on the basis of age or underlying cardiovascular disease, we found an association between traffic-related particles and ST-segment depression that may represent ischemia or myocardial inflammation.


Treating anemia associated with chemotherapy and many cancers is often necessary. However, patient satisfaction with anemia treatment is limited by the lack of validated instruments. We developed and validated a new treatment-specific patient satisfaction instrument: the Patient Satisfaction Questionnaire for Anemia Treatment (PSQ-An). Treatment burden and overall satisfaction scales were designed for ease of use in clinical practice.312 cancer patients  were targeted to complete the PSQ-An at 4 week intervals. Data from weeks 5 and 9 were analyzed. Patients also completed the MOS SF-36 Global Health assessment and questions concerning resources devoted to anemia treatment. Item reduction used endorsement rates, floor/ceiling effects, and item-item correlations. Factor analysis identified meaningful subscales. Test-retest reliability was assessed. Construct validity was tested, using Pearson's correlations, by comparing subscale scores to Global Health, hemoglobin levels, and resources devoted to anemia treatment.P = 0.006).The overall response rate was 92.9% (264/284) at week 5. Most (84.2%) of the patients were female, and the mean (SD) age was 60.2 (± 11.8) years. Two distinct subscales were identified measuring treatment burden (7 items) and overall satisfaction (2 items). Test-retest reliability was examined (ICC: 0.45–0.67); both were internally consistent . Both subscales exhibited convergent and divergent validity with independent measures of health. ANOVA results indicated that the PSQ-An Satisfaction subscale discriminated between 5 levels of MOS SF-36 Global Health (The PSQ-An is a validated, treatment-specific instrument for measuring satisfaction with anemia treatment for cancer patients. PSQ-An subscales reflect the burden of injection anemia treatment on cancer patients and their assessment of the overall treatment value. Anemia and subsequent fatigue have long been recognized as common side effects of cancer itself and its treatments ,2. DepenFurthermore, the presence of anemia is associated with decreased health-related quality of life (HRQL). Holzner  found a Satisfaction with treatment is an important, but poorly studied, aspect of the quality of treatment in supportive oncology care. However, despite the high prevalence of anemia and the growing recognition of treating anemia in cancer patients, there is no assessment tool for evaluating cancer patients' satisfaction with anemia treatment. Defined as a patient-reported assessment of receiving treatment and the outcomes of treatment , treatmeWe developed and validated the Patient Satisfaction Questionnaire for Anemia Treatment (PSQ-An), a disease- and treatment-specific instrument for measuring satisfaction with anemia treatment for cancer patients. The instrument addresses the gap in treatment evaluation tools for oncology care. The PSQ-An was designed to include domains that capture patients' satisfaction with the treatment itself and to include domains pertaining directly to anemia treatment: patient's general satisfaction with treatment, convenience of treatment for patient and family/friends, patients' pain and discomfort, and financial aspects of treatment for the patient. This study reports on the development and validity testing of the scale part of the PSQ-An instrument. Since most enrolled patients were women due to inclusion criteria, this effort should be viewed as an initial validation of the tool; as with most PRO measures, further validation in other patient populations is warranted.The study protocols were approved by the Institutional Review Boards of participating medical centers, and all patients provided written informed consent before any study-related procedures were performed. Patients in the 3 trials were required to have a diagnosis of breast cancer, non-small cell lung cancer , or gynecologic carcinoma of the ovary, cervix, or uterus. Additional inclusion criteria were the following: ≥ 18 years old, anemic (Hb <11 g/dL at screening), expecting to receive ≥ 8 additional weeks of chemotherapy, a Karnofsky performance scale score ≥ 50%; adequate renal function (serum creatinine concentration ≤2.0 mg/dL), adequate liver function , and able to complete questionnaires. Patients were excluded from the trials if they had received a red blood cell transfusion within 4 weeks of screening, or erythropoietic therapy within 2 weeks of randomization; had inadequate iron stores (transferrin saturation < 15% and ferritin < 10 ng/L); known positive antibody response to any erythropoietic agent; known history of pure red cell aplasia, of anemia due to hematologic disorders other than chemotherapy-induced anemia, or of uncontrolled hypertension.The components of the PSQ-An were drawn from other patient satisfaction instruments  for otheThe study sample consisted of 312 adult, English-speaking patients participating in 3 randomized, multicenter trials. This sample size ensures a precision of <5% in the standard errors assuming treatment compliance rates of better than 70%. For logistical and administrative reasons, 3 identical but separate protocols were used, 1 for each tumor type  with a preplanned analysis of all individual data across studies prespecified in each protocol. Patients with breast cancer (n = 141), gynecological malignancies (n = 69), or non-small cell lung cancer (n = 102) were enrolled in the study and were randomized to treatment with darbepoetin alfa or epoetin alfa for anemia due to chemotherapy. The inclusion of patients with 3 different tumor types reduces the likelihood that treatment satisfaction responses are unique to a single population of cancer patients. Following a 1-week screening period, complete blood counts (including Hb) were measured every 2 weeks prior to dosing. In addition, the 4-week recall patient satisfaction questionnaire was administered at weeks 5, 9, 13, and 17.Karnofsky Performance Status ratings  were collected in the trial and converted to Eastern Cooperative Oncology Group (ECOG) Performance Status Ratings to reduce the number of categories with very small numbers of patients used in this analysis. The ECOG Performance Status Rating measures how cancer affects the daily living abilities of the patient . The scaThe 1-item self-report Global Health question from the MOS SF-36 was included  with a 5-point Likert Scale, where a higher score represented better health.The items in the scale part of the instrument originally had response values of 0 to 4 . Values of the 7 negatively stated questions  were reverse scored, so that higher values indicate more positive satisfaction. Items were considered for deletion if they met any of the following 3 criteria: 1) missing responses greater than 10% (endorsement rate); 2) more than 50% of participants reporting either the highest or lowest score available (floor/ceiling effect); or 3) significant item-item correlations ≥ 0.70 . All anaA priori instrument reliability criteria included: 1) item correlation ≥ 0.40 with the total questionnaire  [Principal component analysis was used to identify meaningful and interpretable factors. The number of factors to retain was based on eigenvalues ≥ 1, with factor loadings serving as an indicator of the degree to which each item was associated with each factor. Items were retained in a given factor if they had a factor loading ≥ 0.40. Multi-trait scaling was carried out to evaluate item convergence within scales and item discrimination across scales. istency) ,31, and istency) .Test-retest reliability or reproducibility was assessed using the intraclass correlation coefficients (ICC) . ResponsConvergent and divergent validity were examined by estimating Pearson's correlation coefficient and Spearman's rank-order correlation coefficient, between subscales of the PSQ-An and the MOS SF-36 Global Health, Hb level, and measures of time devoted to treatment hypothesized to assess either similar or different constructs -37. We hDiscriminant validity was assessed by relating PSQ-An subscale factor scores to 3 variables measuring different aspects of patient health: MOS SF-36 Global Health score, Hb level, and ECOG scores. Mean scores on the subscales of the PSQ-An were compared across response categories of the 3 known measures using analyses of variance (ANOVA) . ResponsStatistical analyses were performed using SAS version 8.2 for UNIX .The flow of patients initially enrolled in the study is depicted in Figure Response rates on all PSQ-An question items were greater than 90% Table . Mean, sP < 0.001). Overall satisfaction and likelihood of recommending treatment had r = 0.77 (P < 0.001). All 3 items were retained since the correlations were at or just above the predetermined threshold for consideration and we felt each item measured distinct aspects of treatment burden and satisfaction.Two item-item pairs exhibited correlation coefficients near or greater than 0.70 Table . InterfeP < 0.001). Test-retest reliability was moderate between weeks 5 and 9 in patients with stable MOS SF-36 Global Health scores (128/284 participants). The item ("relating to difficulty in receiving every injection") did not load strongly on either subscale and was excluded from further analyses.Two subscales were identified by factor analysis Table . The firThe Inconvenience subscale correlated negatively with descriptive questions on resources devoted to treatment (Table P = 0.006) relationship with Global Health.The 2 PSQ-An subscales correlated moderately with MOS SF-36 Global Health score, Hb level, and ECOG scores  for measuring satisfaction with anemia injection treatment for cancer patients. Item-item correlations were moderate and suggested that the individual question items measured distinct constructs. There were moderate ceiling effects on several component question items, perhaps reflecting difficulty in measuring high levels of satisfaction. This effect lowered variance in the PSQ-An subscales, which in turn may have led to the moderate results seen in some of the validity tests. For example, the moderate trends noted in convergent, divergent, and known-group discriminant validity may have been due, in part, to limited variation in the subscales. With 40% or more patients reporting high satisfaction on most question items, the potential strength of trends with independent measure of health status are in turn reduced.Items loaded distinctly onto the 2 subscales and internal consistency of both the Inconvenience and Satisfaction subscales were high. Despite including only patients with stable MOS SF-36 Global Health for the test-retest assessment, the subscale scores of the PSQ-An had moderate reproducibility over a 4-week test-retest timeframe (ICC = 0.45 for Satisfaction and 0.67 for Inconvenience). These results were not unexpected given the potential improvement in anemia over 4 weeks of treatment. This suggests that overall satisfaction with treatment may change substantially over the 4-week study period even when controlling for overall health status. Indeed, the effect size for the Satisfaction subscale shows that patients' value assessments of the treatment underwent large changes over the study period.This study has several limitations. First, this study included primarily female patients due to the inclusion criteria, so further evaluation of the PSQ-An is warranted prior to use in other patient populations. Second, the initial item pool was drawn from the literature, not developed from patient focus groups. Nor were these items cognitively tested in patient focus groups. Input from patients may have revealed additional concepts of satisfaction not incorporated into current literature on which our item pool was based. Cognitive testing or debriefing may also have improved the wording/content of the questionnaire; for example, it may have identified better response scales with less potential for ceiling effects. Third, we did not stratify our validation and analysis by disease stage. It is possible that patients with stage IV cancers (48% of our sample) respond quite differently to treatment satisfaction questions than do patients with lesser progression. Again, extrapolations to other patient populations should be made cautiously. Finally, while it is unlikely that the observed ceiling effects are due to response bias, in its present form the PSQ-An may not fully capture the range of satisfaction cancer patients can express about anemia treatment. Consideration could be given to additional response categories to encompass a broader spectrum of satisfaction responses.The Patient Satisfaction Questionnaire for Anemia Treatment (PSQ-An) is a validated, treatment-specific instrument for measuring satisfaction with anemia treatment for cancer patients. The 2 subscales of the PSQ-An reflect the burden of injection anemia treatment on cancer patients and their assessment of the overall value of that treatment. This instrument has potential to aid clinicians in their understanding of the various aspects of patient satisfaction with anemia treatment and allow clinicians to optimize patient care.Robert J. Nordyke is employed by Cerner Health Insights, which provides consulting services to Amgen, Inc. Chiun-Fang Chiou, Joel F. Wallace, and Bin Yao are employed by Amgen, Inc.Robert J. Nordyke: contributed to design, analysis and interpretation of data; drafted the manuscript; revised manuscript for important content; gave final approval of manuscriptChih-Hung Chang: contributed to design, analysis and interpretation of data; drafted the manuscript; revised manuscript for important content; gave final approval of manuscriptChiun-Fang Chiou: contributed to design, analysis and interpretation of data; revised manuscript for important content; gave final approval of manuscriptJoel F. Wallace: contributed to design, collection, analysis and interpretation of data; revised manuscript for important content; gave final approval of manuscriptBin Yao: contributed to design, collection, analysis and interpretation of data; revised manuscript for important content; gave final approval of manuscriptLee S. Schwartzberg: contributed to the interpretation of data; revised manuscript for important content; gave final approval of manuscriptNordyke additional. Appendix: Patient Satisfaction Questionnaire for Anemia Injection Treatment (PSQ-An).Click here for file


Partial or complete seroreversion for HIV-1, or incomplete antibody evolution are relatively rare events that have so far only been described in patients treated with HAART early after virus infection. Whether seroreversion is seen in patients treated effectively with HAART years after their acute infection has not been investigated so far. Therefore we have investigated anti-HIV antibody levels in 80 patients treated with HAART during chronic HIV-1 infection, who had an undetectable HIV-1 plasma viral load for at least five years. In none of the patients we observed seroreversion, and there was also no significant decrease or increase in antibody levels in this group of patients. So, successful HAART treatment during chronic HIV-1 infection does not induce seroreversion. Seroreversion, defined as a quantitative decrease in specific antibody levels so that they measure below the cutoff of an assay, can be partial, resulting in the loss of response against one or a few antigens, or complete, with loss of total antibody reactivity. In HCV infection, seroreversion, which is found in 16–23% of the patients, has been associated with virus clearance, but it can also be observed in chronic HCV infection -6, serorE. coli and B. megaterium), HIV-1 p24 (expressed in E. coli), and synthetic peptides from HIV-1 gp41 and HIV-2 gp36. This assay is available as a qualitative test, but is in fact a kinetic assay whereby increasing amounts of a product are formed with time, and are monitored at multiple points , suggesting it can be used as a semi-quantitative analysis.To examine the effect of long-term undetectable plasma HIV-1 levels upon the serological response, we have analysed the HIV-1/HIV-2 antibody levels in 80 patients treated with HAART resulting in at least a five years undetectable plasma viral load (<50 copies/ml) without blips > 400 copies/ml. Forty-four patients (55%) had an undetectable HIV-1 load for more than 7 years. Fifty patients showed one or two blips of ≤ 400 copies/ml during these years, thirty patients experienced no blips. Presumably, patients with blips have a higher mean residual viremia than patients without blips . Plasma All patients tested HIV seropositive at baseline and after at least five years of undetectable plasma HIV-1 load, irrespective of the occurrence of blips. Table From this study it is clear that seroreversion is not common in HIV infection, not even after the achievement of prolonged low plasma HIV-1 RNA levels, as it was not seen in our group of 80 patients with long-term undetectable HIV-1 load. Earlier observations on HIV seroreversion suggested that seroreversion could occur when HAART is given during acute infection. Our study suggests that HAART given during chronic infection does not induce seroreversion. As seroreversion has been associated with viral clearance, its absence during successful HAART treatment possibly reflects the low level HIV-1 replication in these patients.The author(s) declare that they have no competing interests.HCV: hepatitis C virus. HIV: human immunodeficiency virus. AIDS: acquired immunodeficiency syndrome. HAART: highly active antiretroviral therapy. ELISA: enzyme-linked immunosorbent assay.MC conceived of the study, and participated in its design and coordination, SJ carried out the immunoassays and participated in the design of the study, JMP is the treating physician who collected the samples, MB selected the patients for the study, and ACvdK participated in the design of the study, carried out the statistical analyses and drafted the manuscript. All authors read and approved the final manuscript.


This has been termed force suppression and been found to correlate with ser16-phosphorylation of heat shock protein 20 (HSP20). We tested for the existence of this mechanism in a smooth muscle that is not responsive to nitric oxide.Nitroglycerin can induce relaxation of swine carotid artery without sustained reductions in [CaIsometrically mounted mucosa free rabbit bladder strips were contracted with carbachol and relaxed with 8-Br-cGMP, forskolin, or isoprenaline.Contraction was associated with a highly cooperative relation between MRLC phosphorylation and force such that very small increases in MRLC phosphorylation induced large increases in force. Relaxation induced by 8-Br-cGMP, forskolin, or isoprenaline did not shift the MRLC phosphorylation-force relation from that observed with carbachol alone, i.e. there was no force suppression. HSP20 content was negligible (approximately two hundred-fold less than swine carotid).The lack of force suppression in the absence of HSP20 is consistent with the hypothesized role for HSP20 in the force suppression observed in tonic smooth muscles. Force suppression was found in corpus cavernosum ,5, a NO The author(s) declare that they have no competing interests.TB and JW performed the studies. RM and CR conceived the study. TB and CR drafted the study.


The main limitation in performing genome-wide gene-expression profiling is the assay of low-expression genes. Approaches with high throughput and high sensitivity for assaying low-expression transcripts are urgently needed for functional genomic studies. Combination of the suppressive subtractive hybridization (SSH) and cDNA microarray techniques using the subtracted cDNA clones as probes printed on chips has greatly improved the efficiency for fishing out the differentially expressed clones and has been used before. However, it remains tedious and inefficient sequencing works for identifying genes including the great number of redundancy in the subtracted amplicons, and sacrifices the original advantages of high sensitivity of SSH in profiling low-expression transcriptomes.We modified the previous combination of SSH and microarray methods by directly using the subtracted amplicons as targets to hybridize the pre-made cDNA microarrays (named as "SSH/microarray"). mRNA prepared from three pairs of hepatoma and non-hepatoma liver tissues was subjected to the SSH/microarray assays, as well as directly to regular cDNA microarray assays for comparison. As compared to the original SSH and microarray combination assays, the modified SSH/microarray assays allowed for much easier inspection of the subtraction efficiency and identification of genes in the subtracted amplicons without tedious and inefficient sequencing work. On the other hand, 5015 of the 9376 genes originally filtered out by the regular cDNA microarray assays because of low expression became analyzable by the SSH/microarray assays. Moreover, the SSH/microarray assays detected about ten times more (701 vs. 69) HCC differentially expressed genes (at least a two-fold difference and P < 0.01), particularly for those with rare transcripts, than did the regular cDNA microarray assays. The differential expression was validated in 9 randomly selected genes in 18 pairs of hepatoma/non-hepatoma liver tissues using quantitative RT-PCR. The SSH/microarray approaches resulted in identifying many differentially expressed genes implicated in the regulation of cell cycle, cell death, signal transduction and cell morphogenesis, suggesting the involvement of multi-biological processes in hepato-carcinogenesis.The modified SSH/microarray approach is a simple but high-sensitive and high-efficient tool for differentially profiling the low-expression transcriptomes. It is most adequate for applying to functional genomic studies. Microarray is a powerful technique for simultaneously determining the expression of thousands of genes -3. Such On the other hand, PCR-based suppressive subtractive hybridization (SSH) techniques are highly sensitive for identifying differences in gene expression -9. HowevHerein, we report our modifications using the subtracted amplicons as the targets to hybridize the pre-prepared microarray chips for SSH/microarray analysis. Since all of the probes on the microarray chips have been well characterized, the hundreds and thousands of genes in the subtracted amplicons can be determined by a single hybridization. Moreover, the relative expression status between the compared tissues for each gene can be augmented and easily determined by combining the use of the targets prepared both from forward and reverse subtractions of SSH. We named this modified approach "SSH/microarray" and used human hepatocellular carcinoma as a model to demonstrate the feasibility of this approach.To conduct the comparative transcriptomic studies on human hepatoma particularly for the genes at low-expression levels, we set out an approach as shown in Figure β-actin and GAPDH, with unremarkable difference in hybridization intensities by the regular cDNA microarray assays were efficiently excluded from the subtracted amplicons  Figure  were selSince SSH specifically amplified the difference of gene expression, the SSH/microarray approach should also be able to identify more differentially expressed genes between hepatoma and non-hepatoma liver tissues, which were originally undetectable by the regular cDNA microarray methods. As shown in Fig 2 < 1) using the cDNA microarray assays had a ratio greater than 2 folds (log2 > 1 or < -1) using the SSH/microarray assays  by the regular cDNA microarray assays and by the SSH/microarray assays, respectively . This approach allowed us not only to readily identify genes without tremendous sequencing works in the subtracted amplicons regardless of many redundant clones, but also to easily evaluate the subtraction efficiency and specificity. Moreover, the SSH/microarray approach made it possible to conduct a transcriptome-wide identification of differentially expressed genes particularly for those with low expression. It has been reported that the absolute expression level is not a crucial determinant for identifying genes, while the relative difference in expression levels does impact on whether or not a gene is recovered by subtractive hybridization ,24. In tGBA, PTPRF, GNL3, ERBB3, OGDH, FMO3) were identified to be differentially expressed only by the SSH/microarray assays, but not the conventional cDNA microarrays assaysOne of the main concerns about SSH techniques is their specificity. However, as combined with microarray techniques, the subtraction efficiency can be readily evaluated. As shown in Figure WT1, NME2, IGF2, PPMIG, GMNN, PSMD2, CDK4, SFN, CDC20, CDC25C, AIF1, PINX1, CDC2, MSH2, PA2G4, ASPM, PAFAH1B1, DCTN3) implicated in cell cycle regulation, 16 genes  correlated with the regulation of cell proliferation, 16 genes  involved in the pathways of cell death, 35 genes  related to the regulation of cell differentiation and embryonic development, and 59 genes  associated with signal transduction in response to extra-cellular proliferation and growth stimuli were found. Our findings that differentially expressed genes were related to multi-biological processes suggest the complexity of the molecular mechanisms for hepatocellular carcinogenesis.Of interest, using this novel approach, we identified many of the genes potentially implicated in hepatocellular carcinogenesis, which were not previously identified by conventional cDNA microarray assays. For examples, 18 genes  easy inspection of the subtraction efficiency, 2) avoidance of tremendous sequencing work for gene identification, 3) high sensitivity for identifying the low-expression, differentially expressed genes. This approach allowed for the detection of about ten times more of the differentially expressed genes than did the regular cDNA microarray approach in human hepatoma, particularly for those with low expression. Using this approach, we identified many genes potentially implicated in human hepatocarcinogenesis, which were not identified before. For its high efficiency and high sensitivity, this SSH/microarray approach is powerful for the rapid differentially profiling the low-expression transcriptomes, and most adequate for applying to functional genomic studies.For SSH and cDNA microarray analysis, hepatoma and the corresponding non-cancerous liver tissues were obtained from 3 patients who had liver surgery at the Chang Gung Memorial Hospital. For reverse-transcription real-time PCR, hepatoma and the matched non-hepatoma liver tissues were obtained from additional 18 patients of hepatoma. Diagnoses of HCC and non-hepatoma liver tissues were based on histo-pathologic findings. The Internal Review Board for Medical Ethics of Chang Gung Memorial Hospital approved the specimen collection procedures and informed consent was obtained from each subject or subject's family.Total RNA from hepatoma and the para-hepatoma liver tissues and the poly(A) RNA was prepared as described before ,27. SSH In this study, we used the GMRCL Human 15 K set, Version 2 chips as previously described , which c5-dCTP  using random primers. Unincorporated fluorescent nucleotides were removed using a Qiaquick PCR purification kit (Qiagen). The fluorescent-labeled DNAs were mixed with 30 μg of human cot-1 DNA (Invitrogen) and 100 μg yeast tRNA, precipitated and then resuspended in 30 μl of Microarray Hybridization Buffer Version 2 (Amersham Pharmacia). The hybridization solution was heated to 80°C for 10 min to denature the DNA and was then incubated for 30 min at 37°C, allowing cot-1DNA and yeast tRNA to block the repetitive sequences in genome probes. The probes were hybridized to a human cDNA microarray (GMRCL Human 15 K). We scanned the slides with a confocal scanner ChipReader  and acquired the spot and background intensities with the GenePix Pro 4.1 software . The within-slide normalization was done using programs written with MATLAB 6.5 software .For the SSH experiment, 1 μl subtractive PCR products were labeled with Cy3 and Cy To validate the results obtained from the SSH/microarray assays, we randomly selected six differentially expressed genes identified only by the SSH/microarray assays and three differentially expressed genes identified by both the regular cDNA microarray and SSH/microarray assays for the comparison of gene expression between hepatoma and the corresponding non-hepatoma liver tissues in eighteen patients of HCC using reverse transcription real-time PCR (qRT-PCR). Total RNA was extracted from tissues with Trizol reagents and reverse transcribed using the SuperScript III first strand synthesis system . qRT-PCR was conducted using the ABI PRISM 7000 sequence detection system (Applied Biosystems). Pre-designed Assays on Demand TaqMan probes and primer pairs for these 9 genes were obtained from Applied Biosystems Incoporated (ABI) . For each gene, two to four sets of Taq-Man probes and primers were tested. The probes contained a 6-carboxy-fluorescein phosphoramidite (FAM dye) label at the 5' end of the gene and a minor groove binder and non-fluorescent quencher at the 3' end. These were designed to hybridize across exon junctions. As a result, no fluorescent signal was generated by these assays when genomic DNA was used as a substrate, which confirmed that the assays measured only mRNA. Equal amounts of RNA were used for all qRT-PCR reactions, which were performed in triplicate, and 18S ribosomal RNAs were used as internal controls.All data (derived from three pairs of HCC and non-HCC liver tissues in a dye-swapping approach) were filtered so that 446 up-regulated and 255 down-regulated genes in HCC with at least two-fold difference and p value less than 0.01 were included in the further studies. After removal of the un-annotated genes, a total of 360 and 202 HCC up- and down-regulated genes were subjected to the subsequent gene ontology analyses. The two lists of the differentially expressed genes were analyzed using the on-line software FatiGo for comparative gene ontology categories including molecular function, biological process and cellular component . They weSSH, suppressive subtractive hybridization; HCC, hepatocellular carcinomaY-S Pan and Y-L Lee conducted all of the experiments and contributed to data interpretation.Y-S Lee performed data analysis.W-C Lee collected all of the surgical samples.S-Y Hsieh conceived and designed this study and was responsible for the manuscript preparation.All authors read and approved the final manuscript.The list of the 69 and 701 differentially expressed genes. A total of 69 and 701 differentially expressed genes identified via conventional cDNA microarray and the modified SSH/microarray approaches, respectively, are provided .Click here for file


Whereas NIS expression and regulation have been extensively investigated in healthy and neoplastic thyroid and breast tissues, little is known about NIS expression and function along the healthy and diseased gastrointestinal tract.The sodium/iodide symporter (NIS) is a plasma membrane glycoprotein that mediates iodide (IThus, we investigated NIS expression by immunohistochemical analysis in 155 gastrointestinal tissue samples and by immunoblot analysis in 17 gastric tumors from 83 patients.Helicobacter pylori. Significantly, NIS expression was absent in gastric cancer, independently of its histological type. Only focal faint NIS expression was detected in the direct vicinity of gastric tumors, i.e., in the histologically intact mucosa, the expression becoming gradually stronger and linear farther away from the tumor. Barrett mucosa with junctional and fundic-type columnar metaplasia displayed positive NIS staining, whereas Barrett mucosa with intestinal metaplasia was negative. NIS staining was also absent in intestinalized gastric polyps.Regarding the healthy Gl tract, we observed NIS expression exclusively in the basolateral region of the gastric mucin-producing epithelial cells. In gastritis, positive NIS staining was observed in these cells both in the presence and absence of That NIS expression is markedly decreased or absent in case of intestinalization or malignant transformation of the gastric mucosa suggests that NIS may prove to be a significant tumor marker in the diagnosis and prognosis of gastric malignancies and also precancerous lesions such as Barrett mucosa, thus extending the medical significance of NIS beyond thyroid disease. These hormones are vital for the normal development and maturation of the central nervous system in the newborn and for multiple metabolic functions in the adult. I- metabolism in humans appears to have adapted to provide sufficient I- for normal thyroid function in the face of the environmental scarcity of I-. A cornerstone of I- metabolism is active I- uptake in the thyroid, a process mediated by the sodium/iodide symporter (NIS)..-) is an- for thyroid hormone biosynthesis is governed by dietary I- intake, I- absorption, and thyroidal I- uptake. I- is presumed to be absorbed in the small intestine, but neither the anatomical location of its absorption nor its mechanism has been identified. Interestingly, gastric NIS mediates the active transport of I- from the bloodstream to the gastric lumen, i.e., the active secretion of I- into the gastric juice. Secreted I- is then recirculated into the bloodstream when it is absorbed, along with newly ingested dietary I-, in the small intestine. I- is ultimately excreted mainly by the kidneys. The role of secreted I- in the gastric juice is unknown, as is the function of NIS-mediated I- secretion to the saliva in the salivary glands. By contrast, the functional role of NIS-mediated I- translocation in the lactating mammary gland is crucial and very clear: the process results in I- secretion to the milk, thus supplying the anion to the breast-fed newborn for his/her own thyroid hormone biosynthesis for 5 min. Endogenous peroxide activity was blocked with 5% He protein. The iniImmunohistochemical analysis of NIS in the thyroid, salivary glands, and stomach revealed very distinctly in which particular cells NIS is located in each tissue, namely the thyroid epithelial Fig. , salivarWe investigated NIS protein expression by immnunoblot analysis in 17 gastric tumors (3 MALT and 14 adenocarcinomas) and in their corresponding peritumoral normal tissues. Twelve tumors (71%) exhibited no NIS expression and 5 29%) of the tumoral tissues showed significantly decreased NIS expression compared to that of normal gastric mucosa , signet-ring cell n = 3), papillary (n = 1) and Fig. ]. NIS ex, papillaNine out often hyperplastic gastric polyps were NIS-positive Fig. , the onlHelicobacter pylori (Table The samples from patients with gastritis displayed NIS expression independently of the presence (4/6) or absence 2/6) of /6 of Hel- transport has changed considerably since the extensive review published on the topic in 1961 by Brown-Grant. The main vertebrate nonthyroid tissues reported to accumulate I- actively via NIS are the salivary glands, gastric mucosa, lactating mammary gland, placenta, choroid plexus, and ciliary body of the eye[- or other anions transported by NIS in extrathyroidal tissues is unknown. These tissue-specific NIS-mediated transport systems exhibit functional similarities to their thyroid counterpart, such as a Km for I- ranging from 10 to 30 μM and susceptibility to inhibition by perchlorate (CIO4 -). In contrast to TSH-regulated thyroid NIS and lactationally regulated mammary gland NIS, both salivary and gastric NIS are constitutively expressed[et al [et al [The field of extrathyroidal If the eye,4,8,24. expressed,26. NIS expressed,15,27. Al [et al  have rep- (1.00) ≥ SeCN- (0.87) > SCN- (0.34) > CIO3 -(0.12) > NO3 - (0.04)[- or other anions secreted to the lumen of the gastrointestinal system by the constitutively expressed salivary and gastric NIS are unknown. I- concentrated in the gastric juice is reabsorbed in the small intestine and used for thyroid hormone biosynthesis by the thyroid or excreted by glomerular filtration through the kidney. Albeit with a lower affinity than I-, nitrate (NO3 -) is one of the other anions transported by NIS. Nitrate can be reduced to nitrite (NO2 -) by facultative anaerobic bacteria, and it can be acidified in the stomach, generating nitric oxide (NO), which has a strong bactericidal effect[2 - is bactericidal against H. pylori[-), another NIS substrate[H. pylori is frequently found in patients with gastric ulcers, suggesting a possible causative role for this bacteria[3 - secretion occurs in the stomach, NIS function might play a role in containing or preventing H. pylori-linked gastric ulcers or chronic gastritis. However, we observed no changes in NIS expression in samples from patients with gastritis as compared to normal subjects, irrespective of whether H. pylori was present.NIS transports other anions with the following relative apparent affinities: I - (0.04). As indial effect,30. IndeH. pylori; this efsubstrate. H. pylo bacteria. Thus, oNIS expression and function have been investigated in both thyroid and breast cancers. Whereas NIS expression is absent or decreased in 30% of thyroid cancers, NIS is actually overexpressed but not properly targeted to the plasma membrane in the remaining 70%,22. SignWe ascertained NIS expression in gastrointestinal tumors by carrying out immunohistochemical analysis on biopsies obtained from 66 patients, partly during surgical interventions and partly during endoscopical examinations. In addition, 17 surgical samples were investigated by immunoblot analysis. We detected no NIS staining in patients with intestinalization or gastric cancer, indicating that malignant transformation is linked to decreased or suppressed NIS expression. Interestingly, the focal NIS staining observed in the direct vicinity of gastric tumors increased gradually and became linear as we proceeded away from the tumor. This suggests that loss of NIS expression may precede microscopically identifiable morphological changes. One could speculate that NIS expression in the apparently normal gastric mucosa could be used to define the true healthy margin, thus becoming a potentially useful indicator to decrease local/anastomotic recurrences.99mTcO4 -scintigraphic results from 30 years ago[In junctional and fundus-type columnar metaplasia Barrett mucosa, NIS expression was intact; in contrast, in Barret mucosa with intestinalization, NIS expression was absent. A similar phenomenon was observed in hyperplastic gastric polyps with intestinal metaplasia. These data provide the molecular explanation for the years ago-18,20,21Taken together, our findings underscore the prognostic and diagnostic significance of the absence of NIS expression in gastric alterations when intestinalization or cancer occurs. This is especially true in Barrett metaplasia, since the junctional and fundus-type metaplasias, in which NIS is normally expressed, pose a much lower risk of malignant transformation than intestinal metaplasia, which exhibits no NIS expression and gives rise to a higher number of dysplastic alterations and adenocarcinomas even in small  lesions. In concThe author(s) declare that they have no competing interests.OD and NC conceived the study, AA, OD and NC designed the study and drafted the manuscript, OD and NC generated the NIS Abs and established the protocol for NIS immunohistochemistry, AA and ASZ collected the samples and carried out immunohistochemistries, OD and MP performed immunoblots, IW performed pertechnetate imaging. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Developmental dysplasia of the hip (DDH) is a condition in which bone growth irregularities subject articular cartilage to higher mechanical stresses, increase susceptibility to subluxation, and elevate the risk of early osteoarthritis. Study objectives were to calculate three-dimensional cartilage contact stresses and to examine increases of accumulated pressure exposure over a gait cycle that may initiate the osteoarthritic process in the human hip, in the absence of trauma or surgical intervention.Patient-specific, non-linear, contact finite element models, constructed from computed tomography arthrograms using a custom-built meshing program, were subjected to normal gait cycle loads.Peak contact pressures for dysplastic and asymptomatic hips ranged from 3.56 – 9.88 MPa. Spatially discriminatory cumulative contact pressures ranged from 2.45 – 6.62 MPa per gait cycle. Chronic over-pressure doses, for 2 million cycles per year over 20 years, ranged from 0.463 – 5.85 MPa-years using a 2-MPa damage threshold.There were significant differences between the normal control and the asymptomatic hips, and a trend towards significance between the asymptomatic and symptomatic hips of patients afflicted with developmental dysplasia of the hip. The magnitudes of peak cumulative contact pressure differed between apposed articular surfaces. Bone irregularities caused localized pressure elevations and an upward trend between chronic over-pressure exposure and increasing Severin classification. In the absence of trauma or any surgical intervention, patients afflicted with developmental dysplasia of the hip (DDH) tend to develop osteoarthritis (OA) much earlier than the population norm. Over a lifetime, chronic accumulation of locally elevated cartilage stress exposure may mechanically trigger biologic pathways leading to OA.As a major load-bearing joint, the hip must sustain more than three times body weight during normal human gait -3. DDH iDescriptive assessment schemes of degenerative hip disease are generally 4-bin groupings based on apparent radiographic morphology, from normal to bad; Severin (DDH), BThe objective of this study was to build three-dimensional, patient-specific, non-linear, contact finite element (FE) models from computed tomography (CT) arthrograms to calculate continuous gait cycle articular cartilage contact stress exposure that may initiate OA in the human hip. From these three-dimensional contact stresses, the cumulative pressure exposure per gait cycle was calculated, for symptomatic and asymptomatic hips. Individual peak cumulative (per gait cycle) contact pressures were correlated with Severin classification.We have a unique opportunity to investigate the link between abnormal mechanical load and the earlier-than-normal development of osteoarthritis in a natural hip in the absence of any known trauma or surgical intervention. All patients who had been managed with closed reduction for developmental dysplasia of the hip at the University of Iowa Hospital and Clinics between 1940 and 1969 were identified,13. Clin® [A total of six dysplastic and five asymptomatic hips (six patients) were modeled Table . The ave® ,27. For 1) Normal-appearing asymptomatic hips experience near-normal articular cartilage loading; 2) Bone irregularities in incongruous dysplastic hips significantly elevate local articular cartilage stresses; 3) Significant variability exists in chronic cumulative stresses of hips scaled as normal (Severin I).These patients were selected to address three issues: Hip contact pressure distributions from DDH patient models were compared to the 'normal standard' of an age-matched hip from the Visible Human project. Each FECartilage geometries required additional considerations. For example, cartilage of uniform-thickness, generated by simply extruding the bone surface, included all the irregularities of the bone surface. Cartilage surfaces generated from direct outlines of the radio-opaque contrast enhanced cartilage surfaces in CT arthrograms included the flat spots of hip contact areas. These situations were addressed by smoothing the articular cartilage surface toward an ellipsoidal curvature, for both the acetabulum and the femoral head. The resulting variable thickness of cartilage in subsequent patient-specific FE models  was similar to articular cartilage thicknesses observed in the CT slices,34.Individual patient-specific finite element models were meshed with roughly 5,000 and 20,000 hexahedral elements assigned to the acetabulum and femoral head, respectively, and solved using ABAQUS V6.4). The 3-D FE contact models were non-linear, deformable body, large-displacement and frictionless. The irregular femoral head surface was covered with three layers of smooth-surface, variable thickness cartilage . The 1-mm thick subchondral bone  of the acetabular dome was supported by cancellous bone  with a fixed boundary at the medial pelvic wall : cumulative, for an element wasThe per-cycle cumulative contact pressure, Ppi was the pressure on an element during a gait cycle interval, and Δti was the fraction of time for that increment, . The distributions of cumulative pressure (MPa-seconds per cycle) versus contact area (mm2) were also recorded. These new spatially discriminatory measures separate femoral from acetabular articular cartilage exposure locations and magnitudes with respect to the individual bones. To focus on the effects of patient-specific geometries, we assumed a common activity level of two million gait cycles per year, consistent with reports from pedometer monitored patients[ patients. The cumchronic, was calculated asA chronic exposure damage threshold of 2-MPa was chosen for this study. The normal control had no known musculoskeletal or hip problems and, at 1.229 MPa-sec/cycle on the femoral head and 1.145 on the acetabulum ; Δti was the fraction of time that the pressure Pi was acting on the element; N was the number of years for which the chronic over-pressure was being calculated. The digitized measurements on 20-, 30- and 40-year follow-up films indicated no major joint morphology changes with the radiographs taken at the time of CT examination . Therefore, to maintain focus on the effects of patient-specific geometries on chronic cartilage loading, we assumed each model was a reasonable representation of that patient's hip for twenty years from skeletal maturity, and N was set to 20 years. Any contact pressure under the chronic exposure damage threshold was excluded as normal wear and tear (Pchronic = 0).where Independent t-tests were performed to compare the normal control and the asymptomatic hips. Paired t-tests measured the significance of differences between two different populations: the asymptomatic and symptomatic hips, and between the acetabulae and femoral heads of each patient. Statistical parameters included the peak pressure, the peak cumulative pressure, the peak over-pressure dose, and the mean contact area.A coronal cross-section through a patient's dysplastic hip Figure  highlighA chronic exposure damage threshold of 2-MPa was chosen for this study. The cumulative stress per gait cycle and the chronic contact pressure spatial distribution results for patient 6 with a Severin III dysplastic hip and a Severin I asymptomatic hip were detailed for the acetabulum Figure  and the Normal hip contact results for the Visible Human male were trending towards significance or significantly different from the asymptomatic patient hips in all calculations . This corroborates the previous assertions that the contralateral hip in DDH is also altered by the condition. Therefo2) through the midstance of the normal control (2265 mm2), are consistent with literature values of 759 to 2317 mm2 at midstance[There was a significant reduction in contact area from the Visible Human control to the asymptomatic acetabulae and femoral heads. Overall calculated contact areas, ranging from toe-off of a dysplastic patient  lie within the range of reported values, from 1–10 MPa in normal and dysplastic hips,45. PeakStudies have shown that chronic transient pressure elevations on articular cartilage correlate closely with the progression and onset of OA over many years,12. WhilSpatially discriminatory cumulative contact pressures ranged from 2.45 to 6.62 MPa-per-gait-cycle. Chronic over-pressure doses, for 2 million cycles per year over 20 years, ranged from 0.463 to 5.85 MPa-years using a 2-MPa damage threshold. The spatial distributions of elements experiencing cumulative pressures are located within the superior and posterior portions of the acetabulum . A dysplastic acetabulum has the lowest accumulated stress per gait cycle (2.45 MPa-sec/cycle) while an asymptomatic acetabulae records the highest accumulated stress of 5.48 MPa-sec. Dysplastic femoral heads have the lowest (3.37) and highest (6.6 MPa-sec/cycle) cumulative stresses.Bone morphologic changes characteristic of a DDH patient population with a propensity to OA, act as the focal stress concentrators that may be the forerunner in cartilage degeneration. As shown, the femoral head cartilage is much thinner over bone surface irregularities and experiences increased pressure elevations when in contact with its acetabular counterpart. The FE models are realistic variable cartilage thickness 3-dimensional models with patient-specific anatomies derived from CT scans and incorporating pelvic thickness subchondral and cancellous bone to provide a compliant support of the acetabulum. The non-linear, large-displacement FE models, incorporating continuous gait cycle kinematics and kinetics, are able to discriminate not only local pressure elevations spatially, but also discriminate differences between contact pressure elevations on the acetabulum and the femoral head.There are significant differences between the normal hip and the asymptomatic hips and a trend towards significance between the asymptomatic and symptomatic hips of DDH patients. The magnitudes of peak cumulative contact pressure differ between apposed articular surfaces. Bone irregularities cause localized pressure elevations and an upward trend between chronic over-pressure exposure and increasing Severin classification.The author(s) declare that they have no competing interests.MER carried out the finite element analysis. KHS developed the code used to generate the finite element models. NMG oversaw the model analysis and development of the code. DRP conceived the study and participated in its design and coordination. All authors participated in writing the manuscript. All authors read and approved the final manuscript.


Surgical "anatomical" repair is the most frequent technique used to correct mitral regurgitation due to severe myxomatous valve disease. Debate, however, persists on the efficacy of this technique, as well as on the durability of the repaired valve, and on its functioning and hemodynamics under stress conditions. Thus, a basal and Dobutamine echocardiographic (DSE) study was carried out to evaluate these parameters at mid-term follow-up.Twenty patients selected for the study  underwent pre- and post-operative transthoracic echocardiography (TTE) and intra-operative transesophageal echocardiography (TEE). At mid-term follow-up (20 ± 5 months) all patients underwent rest TTE and DSE (3 min. dose increments up to 40 microg/Kg/min protocol). Pre-discharge and one-month TTE showed absence of MR in 11 pts., trivial or mild MR in 9 pts. and normal mitral valve area and gradients. Mid-term TTE showed decrease in left atrial and ventricular dimension, in pulmonary artery pressure (sPAP) and grade of MR. During DSE a significant increase in mitral valve area, maximum and mean gradients, sPAP, heart rate and cardiac output and a decrease in systolic annular diameter and left ventricular volume were found; in 6 pts. a transient left ventricular outflow tract obstruction was observed.Basal and Dobutamine stress echocardiography proved to be valuable tools for evaluation of mid-term results of mitral valve repair. In our study population, the surgical technique employed had a favourable impact on several cardiac parameters, evaluated by these methods. Over the last 30 years many surgical techniques have been developed to correct mitral regurgitation (MR) in degenerative valve disease, to prevent the occurrence of left ventricular outflow tract obstruction (LVOTO) and to ameliorate the function and durability of the repaired mitral valve, particularly in patients with extensive myxomatous degeneration of mitral valve leaflets -4. TransThe purpose of the present study was to analyse mid-term results of mitral valve repair and hemodynamic changes during DSE in patients referred to our Institution with extensive myxomatous degenerative mitral valve with the features of Barlow's disease .Between October 1998 and January 2004, a total of 330 patients underwent mitral valve repair for pure MR at our Institution. Of these, 33 patients (10%) had Barlow's disease [Twenty out of the 33 patients, all living in the region, represent the study population.There were 12 men and 8 women, mean age was 60 ± 9 years (range 54–72 yrs.). At admission 11 patients were in New York Heart Association (NYHA) class II and 9 in class III. All patients underwent basal pre- and post-operative TTE, and, at mid-term follow-up (20 ± 5 months), rest TTE and DSE. All patients underwent pre-operative coronary angiography. All patients gave their informed consent to the study protocol. The research protocol was approved by the locally appointed ethics committee.At admission into hospital, all patients underwent TTE and TEE with a HP Sonos 5500 , using standard protocols (ASE), to evalIntra-operative TEE was performed before cardiopulmonary bypass (CPB) to confirm pre-operative findings and after weaning from CPB to control the competence of the repaired valve, the absence of LVOTO and the adequacy of trans-mitral flow.TTE was performed before discharge to confirm intra-operative findings and to exclude significant pericardial effusion.One month later the 20 patients underwent basal TTE; after a mean of 20 ± 5 months follow-up (range 12–44 months), they underwent basal and Dobutamine TTE . 2D-images and Doppler data were recorded at rest, at each stage, and at 5 min of recovery. The test was discontinued if any of the following end points was met: 1- target heart rate (>85% of maximum predicted); 2- intolerable symptoms (chest pain or progressive dyspnoea); limiting asymptomatic side effects . The following aspects were evaluated in basal conditions and during stress: the competence of mitral valve, maximum and mean mitral valve gradient by the planimetry of the diastolic CW Doppler velocity signal, the mitral valve area by the pressure half-time formula, the annular dimension from parasternal short-axis view , sPAP, systolic and diastolic artery blood pressure , cardiac output , LVEDV, LVESV, EF, the possible presence of LVOTO. Measurements were averaged from 5 beats in sinus rhythm or 7 beats in atrial fibrillation. Images of all echo-Doppler examinations were performed by one skilled echocardiologist and stored on video-tape for subsequent analysis.The mitral valve was repaired during total normothermic CPB, through conventional midline sternotomy. Myocardial protection was achieved by intermittent anterograde warm blood cardioplegia. The mitral valve was exposed by an incision paralleling Waterston's grove. In presence of ruptured chordae of the posterior mitral leaflet quadrangular resection of the flail segment was performed. In absence of any flail area, part or all the middle scallop of the posterior leaflet was excised. The remnant of the posterior leaflet was detached from the annulus up to the trigons and the height reduced by the excision of its base so as to leave a rim no wider than 1 to 1.5 centimeters. The posterior leaflet was then reattached to the annulus using a 4-0 Ethibond running suture so as to narrow the posterior annulus (sliding technique). Continuity of the posterior leaflet was obtained suturing the two edges with 4-0 Ethibond running sutures. Posterior annuloplasty using a segment of a 3 mm ∅ gore-tex graft completed the repair. The anterior leaflet was approached surgically only in presence of a flail area due to ruptured chordae by inserction of gore-tex cordae passed through the papillary muscles and the free margin of the flail leaflet. One patient with associated CAD underwent myocardial revascularization.CPB time was 66 ± 25 min. and aortic cross-clamp was 50 ± 19 min.Continuous variables were expressed as mean ± standard deviation (SD). Differences between continuous variables at baseline and dobutamine stress-echocardiography were evaluated with paired Student's t-test. Discrete variables were summarised by frequency percent and compared with the chi-square test. One-way analysis of variance (ANOVA) for repeated measures, with the Bonferroni correction for multiple testing, was used to measure the variations of the echocardiographic continuous variables measured at baseline, at one month and mid-term follow-up. All analysws were carried out using SPSS 11.0 . A P value of <0.05 was considered as statistically significant.Pre-operative TTE and TEE showed in all 20 patients moderately severe or severe MR. Prolapse of posterior leaflet associated or not with ruptured chordae was responsible for MR in all patients. In three patients ruptured chordae to the anterior leaflet were also present. There was mild left ventricular enlargement  with preserved systolic ventricular function (EF 60 ± 10%) and mild elevated sPAP (42 ± 16 mmHg). The most important pre-operative TTE findings are listed in Table Intraoperative TEE, before CPB, confirmed pre-operative findings. After weaning from CPB, all patients were in sinus rhythm. TEE demonstrated optimal leaflet coaptation, normal diastolic transmitral flow, trivial or absent MR, absent LVOTO. The mean post-operative stay was 8.5 ± 2.5 days. No hospital deaths occurred.At discharge, TTE showed absence of MR in 11 patients (55%) and mild or trivial MR in 9 patients (45%), mean LVEF 50 ± 7%.One-month TTE showed, in comparison with pre-operative data, a decrease in LA diameter, LV diameter, volume, FS, EF, as well as in grade of MR; no change was found in grade of MR in comparison with discharge results Table .At the latest follow-up , all patients were hemodinamically stable, 16 patients in NYHA class I and 4 in class II.TTE findings remained unchanged, with the exception of sPAP, which was significantly reduced to pre-operative values (42 ± 16 vs 30 ± 11 mmHg) , sPAP, HR, CO, whereas systolic annular diameter, LVEDV, LVESV decreased significantly. No significant difference was found among other echocardiographic parameters Table . At peak, sPAP, HMany surgical procedures have been developed over the years to correct valve regurgitation in degenerative mitral valve disease, to extend feasibility of the repair to the most complex lesions and to optimize durability -4. Good TTE and TEE are the most usual and useful tools to evaluate intra- and post-operative results -17, whilOur study enrolled 20 patients with Barlow's disease and significant MR. All patients underwent surgical "anatomical" repair, which was supported by posterior annuloplasty, with a segment of a 3 mm ∅ gore-tex tube. This technique has the purpose of preserving the morphological, non planar configuration of the mitral annulus, maintaining its physiology, respecting anatomical and functional valvular-ventricular interaction and preventing the occurrence of LVOTO.Our results confirmed the efficacy, the stability and durability of mitral valve repair using this technique, as demonstrated by the intra-operative assessment and the one-month and mid-term echocardiographic follow-up. Further support to the good surgical result was the significant improvement in functional class which occurred in all patients. Many published series of follow-up data have shown that mitral valve repair is a safe and reproducible technique, that results are stable, with low risk of re-operation, decrease in LVESD and LVESV and improvement of LV function is achieved in most patients . Moreover, all these studies have demonstrated that together with the elimination of valve regurgitation lower complication rates than with mitral valve replacement is achieved -21.In our study DSE was performed with the aim to evaluate haemodynamic response under stress conditions and elicit the possible dysfunction of the repaired mitral valve. During stress, functional mitral valve area and mean transvalvular gradient increased significantly, as well as sPAP, HR and CO. There was also an increase in annular area, annular diastolic diameter and annular area index, which was not significant. Systolic annular dimension, LVEDV and LVESV decreased significantly. These data, because of the small sample, are not conclusive, but clearly demonstrate evidence of good valve function, not only at rest but also during stress, further supported by a rise in mitral valve gradients and sPAP, as consequence of haemodynamic effects of Dobutamine, which did not reach pathologic levels. Previous study on the normal mitral valve physiology clearly demonstrated that exercise induces increase of transmitral flow by an increase in mean diastolic cross-sectional area . ConsequWith the technique we describe, mitral annular dynamics have been preserved as demonstrated by the mild, but not significant, increase in diastolic and significant decrease in systolic diameters and by the increase of annular area and annular area index.During DSE only one patient showed trivial MR which was absent at rest and six patients showed transient LVOTO: two patients with intra-ventricular peak gradient < 40 mmHg, two between 40 and 100 mmHg and two > 100 mmHg. The appearance of LVOTO during DSE in non-operated patients was first described in 1992 . A statiA limitation of the present study is the relatively small sample size (only 20 patients); therefore the statistical analysis could have been adversely influenced by inappropriate study sample.A long-term clinical and echocardiographic follow-up is necessary to confirm the stability of the repaired valve.Transthoracic and transesophageal echocardiography have a strategic role in pre-, intra- and post-operative assessment of the mitral valve, particularly in patients with Barlow's disease. In the present study, TTE and DSE proved to be valuable tools for evaluation of mid-term results of "anatomical" repair of MR due to extensive myxomatous degeneration of both leaflets.The surgical technique employed had a favourable impact on several parameters. The majority of patients showed competent mitral valve or mild-trivial MR, with no pathological modification under stress conditions. Furthermore, remarkable reduction in LA and LV dimension was clearly demonstrated at follow-up.