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Visual performances of honeybees have been extensively studied using free-flying individuals trained to choose visual stimuli paired with sucrose reward. By contrast, harnessed bees in the laboratory were not thought to be capable of learning a Pavlovian association between a visual stimulus (CS) and sucrose reward (US). For reasons as yet unknown, harnessed bees only learn visual cues in association with sucrose if their antennae are ablated. However, slow acquisition and low retention performances are obtained in this case. Here, we established a novel visual conditioning protocol, which allows studying visual learning and memory in intact harnessed bees in the laboratory. This protocol consists of conditioning the sting extension reflex (SER) by pairing a visual stimulus (CS+) with an electric shock punishment (US), and a different visual stimulus (CS-) with the absence of shock. Bees with intact antennae learned the discrimination between CS+ and CS- by using chromatic cues, achromatic cues or both. Antennae ablation was not only unnecessary for learning to occur but it even impaired visual SER conditioning because of a concomitant reduction of responsiveness to the electric shock. We thus established the first visual conditioning protocol on harnessed honeybees that does not require injuring the experimental subjects. This novel experimental approach opens new doors for accessing the neural correlates of visual learning and memory in honeybees.

Background: In breast reconstruction, complementary surgery on the contralateral breast is sometimes necessary to obtain a satisfactory aesthetic result. This complementary mammoplasty for symmetry gives the surgeon the opportunity to verify the state of the mammary glandular tissue to rule out a possible occult tumour in the contralateral breast. Our objective was to determine the prevalence of borderline lesions and of in situ and invasive carcinoma in specimens of the contralateral breast in a mammoplasty for symmetry in patients with breast cancer., Methods: We conducted a retrospective study of 145 breast reconstructions with mammoplasty for symmetry conducted at the Tours Regional Teaching Hospital in France., Results: The glandular histologic result after mammoplasty was normal in 45.5% of patients, with benign pathologies in 38.9% and borderline lesions in 15.6% of patients. No invasive or in situ carcinoma was detected., Conclusion: Systematic histologic analysis of glandular mammary tissue sampled after reduction mammoplasty in the particular context of breast reconstruction after breast cancer makes it possible to discover lesions that were not seen in presurgical evaluation. The early management of these borderline occult lesions could reduce the incidence of breast cancer in these at-risk patients.

Background: Aortic regurgitation (AR) is a chronic disease for which there is currently no approved medical treatment. We previously reported in an animal model that β-blockade with metoprolol exerted beneficial effects on left ventricular remodeling and survival. Despite the recent publication of promising human data, β-blockade in chronic AR remains controversial. More data are needed to support this potentially new treatment strategy. We hypothesized that carvedilol might be another safe treatment option in chronic AR, considering its combined β-blocking and α-blocking effects and proven efficacy in patients with established heart failure., Methods and Results: The effects of a 6-month treatment with carvedilol 30 mg/kg/d orally were evaluated in adult Wistar rats with severe AR. Sham-operated and untreated AR animals were used as controls. Carvedilol treatment resulted in less left ventricular hypertrophy and dilatation. Ejection fraction was improved and filling pressures were reduced by carvedilol. β1-Receptor expression was also improved as well as myocardial capillary density. Those beneficial effects were noted despite the presence of drug-induced bradycardia., Conclusions: Carvedilol exerted protective effects against volume-overload cardiomyopathy in this model of aortic valve regurgitation with preserved ejection fraction. These results suggest a protective class effect of β-blockers. Combined with the recent publication of promising human data, our findings support the need to carefully design a prospective study in humans to evaluate the effects of β-blockers in chronic aortic valve regurgitation.

Plasticity in the honeybee brain has been studied using the appetitive olfactory conditioning of the proboscis extension reflex, in which a bee learns the association between an odor and a sucrose reward. In this framework, coupling behavioral measurements of proboscis extension and invasive recordings of neural activity has been difficult because proboscis movements usually introduce brain movements that affect physiological preparations. Here we took advantage of a new conditioning protocol, the aversive olfactory conditioning of the sting extension reflex, which does not generate this problem. We achieved the first simultaneous recordings of conditioned sting extension responses and calcium imaging of antennal lobe activity, thus revealing on-line processing of olfactory information during conditioning trials. Based on behavioral output we distinguished learners and non-learners and analyzed possible learning-dependent changes in antennal lobe activity. We did not find differences between glomerular responses to the CS+ and the CS- in learners. Unexpectedly, we found that during conditioning trials non-learners exhibited a progressive decrease in physiological responses to odors, irrespective of their valence. This effect could neither be attributed to a fitness problem nor to abnormal dye bleaching. We discuss the absence of learning-induced changes in the antennal lobe of learners and the decrease in calcium responses found in non-learners. Further studies will have to extend the search for functional plasticity related to aversive learning to other brain areas and to look on a broader range of temporal scales.

Harnessed bees learn to associate an odorant with an electric shock so that afterward the odorant alone elicits the sting extension response (SER). We studied the dependency of retention on interstimulus interval (ISI), intertrial interval (ITI), and number of conditioning trials in the framework of olfactory SER conditioning. Forward ISIs (conditioned stimulus [CS] before unconditioned stimulus [US]) supported higher retention than a backward one (US before CS) with an optimum around 3 sec. Spaced trials (ITI 10 min) supported higher retention than massed trials (ITI 1 min) and led to the formation of a late long-term memory (l-LTM) that depended on protein synthesis. Our results reaffirm olfactory SER conditioning as a reliable tool for the study of learning and memory.

Background: Exercise training has beneficial effects in patients with heart failure, although there is still no clear evidence that it may impact on their survival. There are no data regarding the effects of exercise in subjects with chronic left ventricular (LV) volume overload. Using a rat model of severe aortic valve regurgitation (AR), we studied the effects of long-term exercise training on survival, development of heart failure, and LV myocardial remodeling., Methods and Results: One hundred sixty male adult rats were divided in 3 groups: sham sedentary (n=40), AR sedentary (n=80), and AR trained (n=40). Training consisted in treadmill running for up to 30 minutes, 5 times per week for 9 months, at a maximal speed of 20 m/minute. All sham-operated animals survived the entire course of the protocol. After 9 months, 65% of trained animals were alive compared with 46% of sedentary ones (P=0.05). Ejection fractions remained in the normal range (all above 60%) and LV masses between AR groups were similar. There was significantly less LV fibrosis in the trained group and lower LV filling pressures and improved echocardiographic diastolic parameters. Heart rate variability was also improved by exercise., Conclusions: Our data show that moderate endurance training is safe, does not increase the rate of developing heart failure, and most importantly, improves survival in this animal model of chronic LV volume overload. Exercise improved LV diastolic function, heart rate variability, and reduced myocardial fibrosis.

Background and Aim of the Study: The calcification of cardiac valves is more frequently observed on left-sided (aortic or mitral) than right-sided (pulmonic or tricuspid) valves. The cause of this preferential left-sided calcification remains relatively unknown. The study aim was to evaluate the capacity of interstitial cells isolated from the four cardiac valves of healthy adult pigs to calcify in culture., Methods: Interstitial cells were isolated from the valve leaflets of three healthy young pigs and cultured in DMEM/fetal bovine serum (10%) in the presence or absence of osteogenic additives (ascorbic acid, dexamethasone, beta-glycerophosphate)., Results: The proliferation rate was similar for cells from each of the four valves. After longer periods of culture (> 10 days), cells from each valve spontaneously formed several calcification nodules, the process being accelerated in the presence of osteogenic additives (to 4-7 days). Alkaline phosphatase (AP) activity was highest in cells originating from the aortic and mitral valves, respectively, and least in those from the pulmonic and tricuspid valves. Culture with the osteogenic additives increased the AP activity by at least 50% for each valve, but the relative AP activity between cells from each valve origin tended to remain similar (aortic > mitral > pulmonic > tricuspid). Interestingly, the levels of matrix Gla-protein mRNA (an endogenous calcification inhibitor) followed an opposite trend of expression for each valve., Conclusion: Interstitial cells from porcine cardiac valves share similarities, although the capacity to calcify is more evident in cells from valves of the left side of the heart. Interstitial cells from the aortic valve displayed the greatest potential for calcification.

Background: Aortic valve regurgitation (AR) imposes a pathologic volume overload to the left ventricle (LV), whereas aerobic exercise causes physiologic volume overloading. The impact of combining both LV volume overloads (pathologic and physiologic) is unknown. Considering the known beneficial effects of aerobic training on the cardiovascular system, we hypothesized that the positive effects would outweigh the negative ones and that exercise would improve the tolerance of the LV to AR., Methods: Forty female adult Wistar rats were randomly divided in the following groups: 1) sham sedentary (SS), 2) sham trained (ST), 3) AR sedentary (ARS), and 4) AR trained (ART). Training consisted in treadmill running for 30 min five times per week at 20 m x s(-1) for 24 wk. In vivo follow-up was made by echocardiography and invasive intracardiac pressure measurements. Hearts were harvested for tissue analysis., Results: Echocardiography revealed less LV dilation and hypertrophy in ART versus ARS as well as improved myocardial performance index. LV ejection fractions remained similar and within normal range in ART versus ARS. Invasive cardiac pressures yielded improved dP/dt- in ART versus ARS but similar dP/dt+. beta(1)-Adrenergic receptor mRNA expression was improved in the ART group versus ARS., Conclusion: Our data suggest that a moderate aerobic exercise program helps minimize LV dilation and hypertrophy and improves diastolic cardiac performance in heart submitted to chronic volume overload due to severe aortic valve regurgitation in this animal model.

Aortic valve regurgitation (AR) imposes a severe volume overload to the left ventricle (LV), which results in dilation, eccentric hypertrophy, and eventually loss of function. Little is known about the impact of AR on LV gene expression. We, therefore, conducted a gene expression profiling study in the LV of rats with acute and severe AR. We identified 64 genes that were specifically upregulated and 29 that were downregulated out of 21,910 genes after 2 wk. Of the upregulated genes, a good proportion was related to the extracellular matrix. We subsequently studied a subset of 19 genes by quantitative RT-PCR (qRT-PCR) to see if the modulation seen in the LV after 2 wk persisted in the chronic phase (after 6 and 12 mo) and found that it did persist. Knowing that the adrenergic and renin-angiotensin systems are overactivated in our animal model, we were interested to see if blocking those systems using metoprolol (25 mg.kg(-1).day(-1)) and captopril (100 mg.kg(-1).day(-1)) would alter the expression of some upregulated LV genes in AR rats after 6 mo. By qRT-PCR, we observed that upregulations of LV mRNA levels encoding for procollagens type I and III, fibronectin, atrial natriuretic peptide, transforming growth factor-beta(2), and connective tissue growth factor were totally or partially reversed by this treatment. These observations provide a molecular rationale for a medical strategy aiming these systems in the medical treatment of AR and expand the paradigm in the study of this form of LV volume overload.

In Pavlovian conditioning, an originally neutral stimulus (conditioned stimulus or CS) gains control over an animal's reflex after its association with a biologically relevant stimulus (unconditioned stimulus or US). As a consequence, a conditioned response is emitted by the animal upon further CS presentations. In such a situation, the subject exhibits a reflex response, so that whether the CS thereby acquires a positive or a negative value for the animal is difficult to assess. In honeybees, Apis mellifera, an odour (CS) can be associated either with sucrose solution (US) in the appetitive conditioning of the proboscis extension reflex (PER), or with an electric shock (US) in the aversive conditioning of the sting extension reflex (SER). The term ;aversive' may not apply to the latter as bees do not suppress SER as a consequence of learning but, on the contrary, start emitting SER to the CS. To determine whether the CS acquires a positive or a negative value in these conditioning forms, we compared the orientation behaviour of freely walking honeybees in an olfactory-cued Y-maze after training them with an odour-sucrose association (PER conditioning) or an odour-shock association (SER conditioning). We show that the same odours can acquire either a positive value when associated to sucrose, or a negative value when associated to an electric shock, as bees respectively approach or avoid the CS in the Y-maze. Importantly, these results clearly establish the aversive nature of SER conditioning in honeybees.

Background: Aortic regurgitation (AR) is a disease of chronic left ventricular (LV) volume overload. Over time, AR will lead to LV dilatation, hypertrophy, and loss of function. There is currently no medical treatment proven effective to slow the evolution of this cardiomyopathy. Vasodilators were once thought to have protective effects, but recent publications have cast some doubts about their effectiveness. We hypothesized that drugs targeting the renin-angiotensin system should be more effective than those having no direct effect on the renin-angiotensin system., Methods and Results: We designed a protocol comparing the effects of 3 vasodilators in a rat AR model (n=9 to 11 animals per group). The effects of a 6-month treatment of (1) nifedipine, (2) captopril, or (3) losartan were compared in male AR rats. Sham-operated and untreated AR animals were used as controls. Nifedipine-treated animals displayed hemodynamics, LV dilatation, hypertrophy, and loss of function similar to those of the untreated group. Both captopril and losartan were effective in improving hemodynamics, slow LV dilatation, hypertrophy, and dysfunction. Gene expression analysis confirmed the lack of effects of the nifedipine treatment at the molecular level., Conclusions: Using an animal model of severe AR, we found that vasodilators targeting the renin-angiotensin system were effective to slow the development of LV remodeling and to preserve LV function. As recently shown in the most recent human clinical trial, nifedipine was totally ineffective. Targeting the renin-angiotensin system seems a promising avenue in the treatment of this disease, and clinical trials should be carefully designed to re-evaluate the effectiveness of angiotensin I-converting enzyme inhibitors or angiotensin II receptor blockers in AR.

Background: The success of social insects can be in part attributed to their division of labor, which has been explained by a response threshold model. This model posits that individuals differ in their response thresholds to task-associated stimuli, so that individuals with lower thresholds specialize in this task. This model is at odds with findings on honeybee behavior as nectar and pollen foragers exhibit different responsiveness to sucrose, with nectar foragers having higher response thresholds to sucrose concentration. Moreover, it has been suggested that sucrose responsiveness correlates with responsiveness to most if not all other stimuli. If this is the case, explaining task specialization and the origins of division of labor on the basis of differences in response thresholds is difficult., Methodology: To compare responsiveness to stimuli presenting clear-cut differences in hedonic value and behavioral contexts, we measured appetitive and aversive responsiveness in the same bees in the laboratory. We quantified proboscis extension responses to increasing sucrose concentrations and sting extension responses to electric shocks of increasing voltage. We analyzed the relationship between aversive responsiveness and aversive olfactory conditioning of the sting extension reflex, and determined how this relationship relates to division of labor., Principal Findings: Sucrose and shock responsiveness measured in the same bees did not correlate, thus suggesting that they correspond to independent behavioral syndromes, a foraging and a defensive one. Bees which were more responsive to shock learned and memorized better aversive associations. Finally, guards were less responsive than nectar foragers to electric shocks, exhibiting higher tolerance to low voltage shocks. Consequently, foragers, which are more sensitive, were the ones learning and memorizing better in aversive conditioning., Conclusions: Our results constitute the first integrative study on how aversive responsiveness affects learning, memory and social organization in honeybees. We suggest that parallel behavioral modules (e.g. appetitive, aversive) coexist within each individual bee and determine its tendency to adopt a given task. This conclusion, which is at odds with a simple threshold model, should open new opportunities for exploring the division of labor in social insects.

Remembering to implement intented activities such as remembering to take medications is referred to as prospective memory (PM). Studies on aging and PM show important variations in the magnitude of the effect of age. One explanation, derived from the multiprocess framework, proposes that age differences depend on retrieval processing. It is thought to be automatic for event-based PM tasks by contrast to self-initiated processing for time-based PM tasks. Older adults generally perform worse than younger ones in laboratory tasks. PM tasks show pronounced age-related deficits, particularly in tasks demanding high level of controlled strategies (e.g. in time-based tasks versus in event-based tasks or, in low salience cue condition versus high salience cue condition in event-based tasks). However, age differences in PM tasks settings differ substantially across studies: some of them show that older adults perform as well as younger ones in event-based PM tasks, especially in ecological ones. Developmental psychologists have investigated this topic, and suggested that several factors could potentially be responsible for the age-related differences observed in MP. These differences could be related to the characteristics of PM cues (e.g. the strength of the association between PM cues and intended actions), the complexity or demands of the ongoing task, or the involvement of planning processes, in other words, the involvement of executive functions. PM deficits are important in mild Alzheimer's disease, even more important than episodic memory or executive functions deficits. This article presents a critical review of cognitive and neuropsychological studies that examined whether these factors or other partly mediate the older adults deficits in prospective memory, which are more important than those observed in episodic memory. The variability of results between studies points to the concept complexity.

Background and Aim of the Study: Chronic aortic regurgitation (AR) induces left ventricular (LV) hypertrophy and eventually LV dysfunction. While the effects of chronic AR on the left ventricle are well known, the effects of acute AR have not been adequately evaluated. It was hypothesized that the LV tissues would be rapidly remodeled by acute AR, and that the renin-angiotensin system would be involved in that acute remodeling., Method: The early LV adaptations to acute AR were evaluated serially over a period of 14 days, using a rat model. Adaptations were evaluated in vivo by echocardiography, and in vitro on explanted heart tissue after one, two, or 14 days., Results: After 14 days, the left ventricle of AR rats was already significantly hypertrophied and dilated (end-diastolic diameter +16% (p <0.05) versus sham; LV mass +16% (p <0.01) versus sham). A short and transient increase in fractional shortening was observed during the first 48 h after AR induction. The cardiomyocyte cross-sectional area and perivascular fibrosis were significantly increased after 14 days of AR. The number of fibronectin-positive cells in LV sections rapidly increased, as did the fibronectin protein and mRNA content of LV crude homogenates. The expression of pro-matrix metalloproteinase 2 was clearly abnormal after two days. Significant shifts in the expression of angiotensin II receptors were also detected as early as one 1 day., Conclusion: Significant macroscopic and microscopic abnormalities were present in the left ventricle of rats with acute AR, soon after its induction. Considerable hypertrophy, perivascular fibrosis and extracellular matrix (ECM) remodeling were present after only 14 days. These results suggest that, in AR, the myocytes and ECM are affected significantly at a very early stage of the disease.

Patients with Alzheimer's disease (AD) show early episodic memory impairments. Such deficits reflect specific impairments affecting one or several stages of encoding, storage and retrieval processes. However, AD patients not only have great difficulty retrieving memories and information but also suffer from distortions of memory, as intrusions and false recognitions. Intrusions can be defined as the unintentional recall of inappropriate information in a laboratory-learning tasks such as word-list recall and story recall. False recognition refers to the erroneous recognition of information that was not previously presented. The first objective of this review is to present studies from the literature that allowed a better understanding of the nature of episodic memory deficits in AD, and to examine recent research on false memories. The second part of this review is aimed at presenting recent research conducted on prospective memory (PM) in Alzheimer's disease. Prospective memory situations involve forming intentions and then realizing those intentions at some appropriate time in the future. Everyday examples of prospective memory include remembering to buy bread on the way home from work, remembering to give friends a message upon next encountering them, and remembering to take medication. Patients suffering from AD show difficulties in performing prospective tasks in daily life, according to the complaints of their care givers, and these difficulties are massively present at the first stages of the disease. Nevertheless, very few studies have been dedicated to this subject, although the evaluation of PM could be helpful for the early diagnosis of AD.

The objective of this study was to assess the long-term effects of beta-blockade on survival and left ventricular (LV) remodeling in rats with aortic valve regurgitation (AR). The pharmacological management of chronic AR remains controversial. No drug has been definitively proven to delay the need for valve replacement or to affect morbidity and/or mortality. Our group has reported that the adrenergic system is activated in an animal model of AR and that adrenergic blockade may help maintain normal LV function. The effects of prolonged treatment with a beta-blocker are unknown. Forty Wistar rats with severe AR were divided into 2 groups of 20 animals each and treated with metoprolol (Met, 25 mg.kg(-1).day(-1)) or left untreated for 1 yr. LV remodeling was evaluated by echocardiography. Survival was assessed by Kaplan-Meir curves. Hearts were harvested for tissue analysis. All Met-treated animals were alive after 6 mo vs. 70% of untreated animals. After 1 yr, 60% of Met-treated animals were alive vs. 35% of untreated animals (P = 0.028). All deaths, except one, were sudden. There were no differences in LV ejection fraction (all >50%) or LV dimensions. LV mass tended to be lower in the Met-treated group. There was less subendocardial fibrosis in this group, as well as lower LV filling pressures (LV end-diastolic pressure). beta-Adrenergic receptor ratio (beta(1)/beta(2)) was improved. One year of treatment with Met was well tolerated. Met improved 1-yr survival, minimized LV hypertrophy, improved LV filling pressures, decreased LV subendocardial fibrosis, and helped restore the beta-adrenergic receptor ratio.

We present the case of a 53-year-old menopaused woman who developed an invasive persistent trophoblastic uterine disease with several lung metastasis. Patient case was listed in a highly risked group in the WHO classification. As the patient refused primary hysterectomy she received polychemotherapy alone. Monitoring of the treatment and vascularity evolution of the tumor was followed through pelvic endovaginal ultrasound using 3D and contrast enhancement as well as HCG decrease in the context of uterus conservation that was wished by the patient.

The early events leading to the establishment of left ventricular hypertrophy associated to pressure overload (PO) are not well characterized. To explore these early events, aortic banding (AB) was performed in rats to induce left ventricle (LV) PO. Animals were sacrificed after 24, 48 h or 14 days. An echocardiogram was performed before the procedure and at sacrifice. LVs were preserved for the evaluation of fibrosis, angiotensin II (AT) receptors expression and stress-related MAP kinases (ERK 1/2, JNK and p38) pathways. We observed that concentric LV hypertrophy was established after only 14 days. Collagen I and fibronectin gene expressions were decreased the first 2 days after AB induction whereas AT receptors mRNA levels were sharply increased. ERK 1/2 and JNK activities in LV homogenates were decreased 24 h after AB but came back to normal after 14 days. p38 activity however was stable during the period studied. We also evaluated the presence of two phosphorylated transcription factors related to JNK signaling pathway (ATF-2 and c-Jun) in cardiomyocyte nuclei. The proportion of LV cell nuclei positive for these two activated transcription factors was significantly reduced in AB rats compared to sham. These results suggest that the early response of the LV to acute PO is to attenuate the expression of some pro-fibrotic and pro-hypertrophic signaling pathways and possibly AT signaling by decreasing ERK 1/2 and JNK relative activities.

Invertebrates have contributed greatly to our understanding of associative learning because they allow learning protocols to be combined with experimental access to the nervous system. The honeybee Apis mellifera constitutes a standard model for the study of appetitive learning and memory since it was shown, almost a century ago, that bees learn to associate different sensory cues with a reward of sugar solution. However, up to now, no study has explored aversive learning in bees in such a way that simultaneous access to its neural bases is granted. Using odorants paired with electric shocks, we conditioned the sting extension reflex, which is exhibited by harnessed bees when subjected to a noxious stimulation. We show that this response can be conditioned so that bees learn to extend their sting in response to the odorant previously punished. Bees also learn to extend the proboscis to one odorant paired with sugar solution and the sting to a different odorant paired with electric shock, thus showing that they can master both appetitive and aversive associations simultaneously. Responding to the appropriate odorant with the appropriate response is possible because two different biogenic amines, octopamine and dopamine subserve appetitive and aversive reinforcement, respectively. While octopamine has been previously shown to substitute for appetitive reinforcement, we demonstrate that blocking of dopaminergic, but not octopaminergic, receptors suppresses aversive learning. Therefore, aversive learning in honeybees can now be accessed both at the behavioral and neural levels, thus opening new research avenues for understanding basic mechanisms of learning and memory.

Background and Aim of the Study: Aortic valve regurgitation (AR) can result in heart failure from chronic overloading of the left ventricle. As little is known of gender-specific responses of the left ventricle to this condition, the study aim was to compare left ventricular (LV) remodeling in male and female rats with severe AR. In order to assess the impact of estrogens on LV remodeling in AR, the effect of ovariectomy (OVX) was also evaluated., Methods: AR was created in adult Wistar rats (females (control or OVX) and males). Animals were followed for 26 weeks and compared to sham-operated groups. Heart function was evaluated in vivo using echocardiography, and the hearts were subsequently harvested for tissue analysis., Results: The LV ejection fraction was decreased similarly in both sexes. Despite similar echocardiographic AR severity, females had higher indexed cardiac output and the largest increase in LV weight, cardiomyocyte hypertrophy and eccentric remodeling. No differences were observed between control and OVX females. Ovariectomy had no significant impact on any of the parameters monitored., Conclusion: Female rats developed more LV remodeling in response to chronic AR than males. AR appears to impose a greater LV workload on females due to their smaller body and heart size. Hormonal status did not have any impact on LV remodeling in this experimental model.

Since the invention of Northern blot profiling more than 30 years ago, scientists have progressed from being able to analyze only a few genes at a time to whole-genome and global differential gene expression profiling. This progress has been made possible by the development of several high-throughput technologies. Such technologies have gained increasing popularity in recent years because of significant discoveries made in the field of proteomics, and have already led to huge advances in the medical field. This article reviews the high-throughput technologies that have been undergoing development during the last two years for the selection of specific cell subtypes in a tissue, for the discovery and identification of all of the genes expressed in a system without pre-selection (open systems), and for the validation of selected genes (closed systems).

Objectives: The purpose of this study was to compare aortic valve function and morphology in adult wild-type (WT) mice and in low-density lipoprotein receptor-deficient (LDLr-/-) mice fed or not fed a high-fat/high-carbohydrate (HF/HC) diet., Background: Observations suggest a link between degenerative aortic valve stenosis (AS) and atherosclerosis. Aortic valve stenosis has been successfully induced in animal models of extreme hypercholesterolemia, but these models are less relevant to humans. It is not known if a proatherogenic HF/HC diet without added cholesterol could have the same negative impacts., Methods: Forty C57BL/6J mice were divided into four groups: WT + normal diet, WT + HF/HC diet, LDLr-/- with a normal diet, and LDLr-/- with a HF/HC diet. Aortic valve function and histology were evaluated by echocardiography after four months., Results: Wild-type mice on a HF/HC diet became mildly hypercholesterolemic, obese, and hyperglycemic. As expected, LDLr-/- mice became severely hypercholesterolemic. Both WT and LDLr-/- mice on a HF/HC diet displayed smaller valve areas and higher transvalvular velocities (p < 0.01) after four months. Aortic valve leaflets were thicker and infiltrated with lipids and macrophages in both HF/HC groups., Conclusions: A HF/HC diet in mice results in significant aortic valve abnormalities. Putting WT mice on a HF/HC diet reproduced a combination of atherogenic factors (obesity, mild dyslipidemia, and hyperglycemia) more commonly encountered in humans than isolated severe hypercholesterolemia. Severe hypercholesterolemia was not a prerequisite in our model. This experimental model suggests that AS development is multifactorial and that hypercholesterolemia should not be the only target in this disease.

Background: Dobutamine stress echocardiography is used to investigate a wide variety of heart diseases in humans. Dobutamine stress echocardiography has also been used in animal models of heart disease despite the facts that the normal response of healthy rat hearts to this type of pharmacological stress testing is unknown. This study was performed to assess this normal response., Methods: 15 normal adult male Wistar rats were evaluated. Increasing doses of dobutamine were infused intravenously under continuous imaging of the heart by a 12 MHz ultrasound probe., Results: Dobutamine stress echocardiography reduced gradually LV diastolic and systolic dimensions. Ejection fraction increased by a mean of +24% vs. baseline. Heart rate increased progressively without reaching a plateau. Changes in LV dimensions and ejection fraction reached a plateau after a mean of 4 minutes at a constant infusion rate., Conclusion: DSE can be easily performed in rats. The normal response is an increase in heart rate and ejection fraction and a decrease in LV dimensions. A plateau in echocardiographic measurements is obtained after 4 minutes of a constant infusion rate in most animals.

Two mixtures of Propionibacterium freudenreichii commercial strains were tested as adjunct cultures in pasteurized milk Raclette cheese to investigate the ability of propionibacteria (PAB) to enhance flavor development. Cheese flavor was assessed by a trained sensory panel, and levels of free amino acids, free fatty acids, and volatile compounds were determined. The PAB level showed a 1.4 log increase within the ripening period (12 weeks at 11 degrees C). Eye formation, which was not desired, was not observed in PAB cheeses. PAB fermented lactate to acetate and propionate and produced fatty acids by lipolysis, branched chain volatile compounds derived from isoleucine and leucine catabolism and some esters. One of the experimental cheeses received the highest scores for odor and flavor intensity and was characterized by higher frequencies of detection for some minor notes ("propionic"and "whey" odor, "sweet" taste). PAB can therefore be considered as potential adjunct cultures to enhance or modify cheese flavor development.

Caveolins are highly expressed in terminally differentiated cells, but this expression is down-regulated in various cancer cell lines. Exposure to low doses of paclitaxel (taxol) is sufficient to up-regulate caveolin-1, suggesting that a mild cytotoxic stress induces a response implying caveolin and caveolae. Here we show that this up-regulation is sustained even after the cessation of paclitaxel treatment. After exposure to a cytostatic dose of paclitaxel (50 nM), A549 lung cancer cells are blocked in the G2/M cell cycle phase. After removal of paclitaxel, cell death occurs, accompanied with an increase in caveolin expression, suggesting an effect of caveolin in this process. Three days post-paclitaxel treatment, surviving A549 cells were passaged and only a half of them adhered to the culture dish. Adhering cells (still mainly in the G2/M cell cycle phase) were still unable to grow and progressively entered in an apoptotic state. This study suggests that effects of a low dose of paclitaxel were still present even 1 week after drug removal and that caveolin-1 is a good marker of cytotoxicity.

Caveolin 1 expression is downregulated in various cancer cell lines. Interestingly, in several drug-resistant cancer cells, a strong induction of caveolin 1 expression has been reported suggesting a role for caveolin 1 in the acquisition and/or the maintenance of multidrug resistance phenotype. In addition, it was reported that p-glycoprotein localized to caveolin-rich membrane domains in these cells. In this study, we progressively exposed A549 lung adenocarcinoma cells to increasing doses of etoposide. Both R1 and R2 cell lines had greatly increased levels of p-glycoprotein expression while mrp expression levels were moderately increased but only R2 cells had raised caveolin levels compared to control A549 cells. Both caveolin-1 and p-glycoprotein colocalize in Triton-insoluble membrane domains in all our cell lines but only caveolins-1 was solubilized by the addition of octylglucoside at 4C suggesting that these two proteins are located in different membrane domains. Using an anti-caveolin-1 antibody, we did not succeed to immunoprecipitate p-glycoprotein. Interestingly, total cellular cholesterol (the major lipid component of caveolae and triton-insoluble domains) was greatly increased in both R1 and R2 cell lines compared to naive A549 cells.

Background: Past studies have suggested that the adrenergic system becomes abnormally activated in chronic volume overload, such as in severe aortic valve regurgitation (AR). However, the effectiveness of agents directed against this adrenergic activation has never been adequately tested in chronic AR. We therefore tested the effects of metoprolol treatment on the left ventricular (LV) function and remodeling in severe chronic AR in rats., Methods and Results: Severe AR was created in adult male Wistar rats by retrograde puncture of the aortic leaflets under echocardiographic guidance. Two weeks later, some animals received metoprolol treatment (25 mg/kg) orally for 24 weeks, and some were left untreated. LV dimensions, ejection fraction, and filling parameters were evaluated by echocardiography. Hearts were harvested at 1, 2, 14, and 180 days for the evaluation of hypertrophy, beta-adrenergic receptor status, and extracellular matrix remodeling. We found that metoprolol treatment prevented LV dilatation and preserved the ejection fraction and filling parameters compared with untreated animals. Metoprolol increased the expression of beta1-adrenoreceptor mRNA and reduced G protein receptor kinase 2 levels. Collagen I and III mRNA levels were reduced. Cardiac myocyte hypertrophy was also prevented., Conclusions: In our experimental model of severe AR, metoprolol treatment had a significant beneficial global effect on LV remodeling and function. These results suggest that the adrenergic system is important in the development of volume-overload cardiomyopathy in AR and that adrenergic-blocking agents may play a role in the treatment of this disease.

The efficacy of angiotensin-converting enzyme inhibitors (ACEIs) in the treatment of chronic aortic regurgitation (AR) is not well established and remains controversial. The mechanisms by which ACEIs may protect against left-ventricular (LV) volume overload are not well understood, and clinical trials performed until now have yielded conflicting results. This study was therefore performed to assess the effectiveness of two different doses of the ACEI captopril in a rat model of chronic AR. We compared the effects of a 6-month low-dose (LD) (25 mg/kg) or higher dose (HD) (75 mg/kg) treatment with captopril on LV function and hypertrophy in Wistar rats with severe AR. Untreated animals developed LV eccentric hypertrophy and systolic dysfunction. LD treatment did not prevent hypertrophy and provided modest protection against systolic dysfunction. HD treatment preserved LV systolic function and dimensions and tended to slow hypertrophy. The cardiac index remained high and similar among all AR groups, treated or not. Tissue renin-angiotensin system (RAS) analysis revealed that ACE activity was increased in the LVs of AR animals and that only HD treatment significantly decreased angiotensin II receptor mRNA levels. Fibronectin expression was increased in the LV or AR animals, but HD treatment almost completely reversed this increase. The ACE inhibitor captopril was effective at high doses in this model of severe AR. These effects might be related to the modulation of tissue RAS and the control of fibrosis.

Caveolin 1 expression is down-regulated in various cancer cell lines. Interestingly, in several drug-resistant cancer cells, a strong induction of caveolin 1 expression has been reported, suggesting a role for caveolin 1 in the acquisition and/or the maintenance of the multidrug-resistance phenotype. Here, we show, in drug-sensitive lung cancer cells (A549, Calu-6 or NCI-H69), that exposure to cytotoxic drugs (taxol, doxorubicin or etoposide) is sufficient to strongly up-regulate caveolin 1 and 2 protein levels. This up-regulation is sustained even 1 week after drug removal. Our results suggest that caveolin up-regulation is an early cellular response to a cytotoxic stress taking place before drug resistance.

We studied the hepatotoxic effect of heavy metals (cadmium, mercury, copper) on Mg2+ -ATPase, NADH diaphorase, succinic dehydrogenase and acid phosphatase of yellow-legged gull liver, using enzyme histochemical methods. The lysosomal enzyme activity of acid phosphatase was increased in all cases. However, the other enzyme activities appeared to be insensitive to the different metallic pollutants and to their respective levels, in contrast with literature experimental data showing plasma membrane and mitochondrial alterations. This controversy could be explained by the differences in dietary conditions and metal overloads. The molecular basis of the toxicities of metallic pollutants is discussed.

Little is known of the mechanisms allowing neutrophils to infiltrate tissue after transendothelial migration. We postulated that VLA6 might be involved in neutrophil infiltration because it revealed as the most expressed beta1 integrins among VLA5, VLA4, and VLA3, which also appeared to define subsets within the blood neutrophil population. Transendothelial migration up-regulated by threefold (5,000 to 15,000 receptors) VLA6 expression on neutrophils. VLA6 up-regulation was transient, peaking in 6 min and returning to baseline in 1 h when tested in response to N-formyl-methionyl-leucyl-phenylalanine. Neutrophil degranulation experiments revealed a steady correlation between expression of VLA6 and granule content release, notably beta-glucuronidase, indicating that VLA6 molecules were preformed and stored mostly in azurophilic granules. Migration across fibroblast monolayers of neutrophils preactivated for VLA6 up-regulation was blocked when they were preincubated with anti-VLA6. However, anti-VLA6 had no effect on transfibroblast migration of non-preactivated neutrophils. These results indicate that VLA6 was functional only on activated neutrophils that used their up-regulated VLA6 to cross fibroblasts. Activation of neutrophils by transendothelial migration induces rapid expression of granule release VLA6 that appears to be a key adhesion mechanism used by a subset of neutrophils to infiltrate tissue.

We investigated whether cytokine genes were activated in human tumour-infiltrating mononuclear leukocytes (TIML) obtained from six lung adenocarcinomas and seven glioblastomas. TIML were extracted by mechanical disruption and isolated by double density gradient of Ficoll. We performed mRNA reverse transcription-polymerase chain reaction (RT-PCR) on these fresh (noncultured) TIML and autologous peripheral blood mononuclear leukocytes (PBML) using primers for the cytokines IL-1 beta, IL-6, IL-2, IL-4, GM-CSF, IFN-gamma and TNF-beta. In addition, we compared patients' TIML and PBML populations with healthy normal and alpha-CD3 activated PBML as an optimally activated reference population. Gel bands of RT-PCR products were quantitated in relative units (RU) as a function of their size and intensity by computerized image-analysis. Lung and brain patients' TIML showed IL-1 beta and IL-6 cytokine mRNA expressed in the average of 2-log RU but not significantly different from autologous and normal healthy PBML. IL-2, IFN-gamma and TNF-beta also did not appear expressed in the TIML at higher levels than in autologous or healthy normal PBML. However in two thirds of patients, lung TIML could be distinguished from autologous PBML by specific expression of GM-CSF and from healthy normal PBML by expression of IL-4. Similarly, most brain TIML expressed mRNA significantly above healthy normal PBML for GM-CSF and IL-4. In comparison with alpha-CD3 activated healthy PBML, our results suggest that lung and brain TIML had detectable cytokine mRNA, but they seemed poorly activated in total number of genes and amount of cytokine mRNA.(ABSTRACT TRUNCATED AT 250 WORDS)

Little is known about the activation level of tumor-infiltrating lymphocytes (TIL) in human lung adenocarcinoma. We investigated the activation of fresh TIL at cellular and molecular levels and compared it with autologous and healthy normal peripheral blood lymphocytes (PBL) for baseline level. TIL were extracted from 12 primary lung adenocarcinomas by mechanical disruption without enzyme use and isolated by double-density Ficoll gradients. Flow-cytometry analysis of TIL subset distribution revealed that the majority was composed of T lymphocytes, and double labeling with alpha-CD3 and adhesion/activation markers revealed T cell subsets expressing CD49a, CD49b, CD54, and CD15, each of which was almost absent in autologous T peripheral blood lymphocytes (T-PBL). Moreover, the proportions of T-TIL expressing CD58, CD65, or CD25 were increased severalfold compared to T-PBL. Lymphokine gene activation in TIL was assessed by mRNA reverse transcriptase/polymerase chain reaction (RT-PCR) and primers for interleukin(IL)-2, IL-4, interferon (IFN) gamma, granulocyte/macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor (TNF) beta. Semiquantitative comparisons between patients' TIL and PBL and healthy normal and activated PBL were performed by computerized image analysis. RT-PCR gel band products were quantified in relative units as a function of their size and intensity. TIL expressed detectable lymphokine mRNA but seemed poorly activated with respect to the total number of lymphokine genes and the amount of mRNA compared with alpha-CD3-activated healthy PBL. IL-2, IFN gamma, and TNF beta did not appear to be expressed at higher levels in TIL than in autologous or healthy normal PBL. However, two-thirds of the patients had TIL distinguishable from autologous PBL by specific expression of GM-CSF and from healthy normal PBL by expression of IL-4. These results show that lung adenocarcinoma TIL populations had little lymphokine gene activation despite the presence of several T cell subsets expressing different adhesion/activation markers. The lack or deficient combination of lymphokine production may be a factor that prevented efficient activation of TIL in these tumors.

Alterations in expression of various cell-adhesion molecules have been reported in a variety of malignant tissues. However, little is known about how lung adenocarcinomas differ in CAM expression from the normal lung. We analyzed the expression of integrins alpha 1 beta 1 through alpha 6 beta 1, intercellular adhesion molecule (ICAM)-1, neural cell adhesion molecule (NCAM), and lymphocyte function antigen (LFA)-3, CD44, and the two carbohydrate antigens, Lewisx (Le(x)) and sialosyl-Le-Le(x) of lung adenocarcinoma cells, and compared them with autologous pneumocytes. CAM expression was studied by an immunohistochemical method using monoclonal antibodies, and computerized image analysis was used to quantify the immunoperoxidase-staining intensity. The normal lung alveolar cells strongly expressed the integrins alpha 1 beta 1 and alpha 3 beta 1, and fairly expressed alpha 2 beta 1, alpha 4 beta 1, alpha 5 beta 1, and alpha 6 beta 1. ICAM-1, LFA-3, and CD44 were strongly expressed, whereas NCAM, the Le(x) and sialosyl-Le-Le(x) antigens, were expressed weakly. In contrast, we did not detect expression of the alpha 1 beta 1 integrin on any autologous lung adenocarcinoma cells, and they showed on average a 50% reduction in labeling relative intensity units for the integrin common chain marker beta 1, the specific integrins alpha 3 beta 1, alpha 5 beta 1, and alpha 6 beta 1, and ICAM-1, and LFA-3. Examination of the adjacent small blood vessel endothelium in malignant lung tissues did not reveal any major alterations in CAM expression, the small vessel endothelium of the normal and malignant lung tissues appeared with a similar CAM profile. These results suggest that lung adenocarcioma cells have a lack of alpha 1 beta 1 expression and significant reduction in some other integrin beta 1 and CAM expression in comparison with their autologous pneumocytes. This aberration in CAM expression by the lung adenocarcinoma cells may be involved in their loss of proliferation control and may interfere with leukocyte adhesion to tumor cells, enabling the tumor to escape immunodestruction.

The authors present a case of telangiectatic osteogenic sarcoma of unusual radiographic presentation. The lesion presents as a slow growing, rather "benign" tumor, while most osteosarcomas usually present as osteolytic lesions of which the radiological aspect allows the diagnosis of malignancy with certainty.

CTL (cytotoxic T lymphocytes) and LGL (large granular lymphocytes) exocytose cytoplasmic granules on activation after recognition of their target, releasing granule-associated molecules. We have previously suggested that this process could release immunoregulatory molecules. In this study we investigated whether normal human LGL granules contained a factor regulating different macrophage activity. Human CD3+ LGL cells were generated by activating peripheral blood lymphocytes (PBL) for 10-12 days with recombinant human IL-2 (rhIL-2), and granules were isolated from disrupted cell homogenate by Percoll gradient fractionation. Solubilized granules were tested for macrophage-activating factor (MAF) activity in three different macrophage assays. When M-CSF-differentiated murine bone marrow-derived macrophages were incubated 9 hr with human LGL granules, they were fully activated to lyse the TNF-resistant P815 tumor cells. The granule-MAF showed a synergistic effect with rhIL-1 beta, rmTNF-alpha, and rmIFN-tau in the cytolytic assay. In addition, proteose-peptone-elicited murine peritoneal macrophages profoundly increased H2O2 production after activation with human LGL granules. However, unlike IFN-tau, no increase in peritoneal macrophage Ia antigen expression was detected after incubation with granules. Moreover, granule-MAF suppressed Ia induction by IFN-tau. These results confirm that human CD3+ LGL granules contain a molecule(s) capable of regulating macrophage function.

It has been reported that lymphocytes from peripheral blood (PBL) cultured with interleukin-2 (IL-2) produce predominantly CD16+ lymphokine-activated killer (LAK) cells. We developed a two-step method to generate LAK cells from human PBL in long-term cultures (10-12 days) with recombinant human IL-2 (rhIL-2) and characterized the evolving LAK cell population by testing its phenotype and cytotoxic activity as a function of time. The starting PBL displayed some natural killer (NK) cytotoxicity but no LAK activity. At day 6, the cells were a mixed population of about 80% CD3+ and 6% CD16+ cells. Little proliferation was evident but strong LAK activity was detected. After 10-12 days, major cell expansion had occurred and they were essentially a pure (greater than 90%) CD3+ CD16- CD56- cell population large granular lymphocyte (LGL) by morphology that displayed strong non-MHC-restricted killing activity (greater than 200 lytic units). Over the same period of time, the CD16+ cells had almost completely regressed in these cultures. This preferential induction of CD+ LAK cells was not an effect of IL-2 concentration as 10 U/ml was as effective as 500 U/ml. Further characterization revealed a major population of CD4+ (60%) and CD8+ (30%) with a smaller fraction (less than 9%) of gamma delta + cells. These results indicate that a virtually pure CD3+ LAK cells population was produced with long-term cultures of lymphocytes from peripheral blood in rhIL-2, in which active proliferation of the CD3+ but not CD16+ cells occurred.

Cancer patients are frequently immunodepressed and this could be related to undernutrition, particularly in gastrointestinal (GI) neoplasia. We therefore looked for correlations between different leucocyte subsets' (T11+, T4+, T8+, B1+, and Mo2+) cells in the peripheral blood of these patients and their nutritional state. Significant alterations were found in absolute number/ml of T11+, T4+, B1+, and Mo2+ cells. Malnourished cancer patients were more affected than those appearing well-nourished, exhibiting a decrease of 50% in the number of T11+, T4+, and B1+ cells as compared with healthy controls. The phytohemagglutinin A (PHA) stimulation index (SI) was also significantly decreased in the malnourished group. Linear regression analysis between SI and these leucocyte subsets revealed that only the T4+ cells offered a significant correlation (r = 0.6169 p less than 0.01). These results suggest that the absolute number/ml of T4+ cells reflects the nutritional state of patients bearing GI tumors.

Recent work from our laboratory has shown that NK cells rapidly release preformed factor(s) that stimulate monocyte oxidative metabolism and microbicidal activity. We have hypothesized that such factors could also activate macrophage (M phi) tumor lysis and might be stored in the cytoplasmic granules. Granules were isolated from the RNK large granular lymphocyte leukemias by nitrogen cavitation and Percoll fractionation of the cell homogenate. Utilizing CSF-1 differentiated murine bone marrow-derived M phi and P815 tumor target cells, a M phi-activating factor (MAF) was found. The MAF activity was identified in two peaks, the first was coincident with dense granule enzymes and was 60 times more concentrated per mg protein than a second peak in the cytosol fractions. Solubilization in 2 M NaCl was necessary to recover activity from both peaks. Granule NK-MAF required the simultaneous presence of LPS in order to induce tumoricidal activity. Kinetics of NK-MAF activation peaked after 12 h of exposure. The NK-MAF was short lived in the solubilized granules; however, its heat resistance allowed us to prepare enriched and stable preparations. Treatment of NK-MAF with pepsin but not trypsin completely abrogated its activity. The NK-MAF passed through an ultrafiltration membrane with a nominal cut-off of 10 kDa. This work indicates that NK cell granules contain a small heat-stable peptide capable of activating M phi tumoricidal activity.