Out of 185 submitted projects, the Institute ranked fifth to eighth, and became Centre of Excellence of European Union. Experts of the European Commission acknowledged the orientation of the project, the significance of the topic, the multidisciplinary approach, the international cooperation involved and the expected positive impact of the results upon the European population. They underlined the characteristics of the Institute as a dynamically developing research facility with broadly established international cooperation, which can stand the proof with institutions in EU countries. One of the chief factors substantiating the selection of the Institute was the international cooperation of the majority of researchers with outstanding laboratories in Europe (Amsterdam, Marseille, Strasbourg, Lund, Budapest, Bristol, Oslo, Graz, etc.), which has resulted in more than 70 joint publications appearing in scientific journals abroad. The positive assessment of the Institute was supported also by the high profile of its research workers: 11 of them hold the highest scientific degree (DSc), the majority are internationally recognized scientists, regularly lecturing abroad, participating in international congresses, often as chairpersons of sections and invited lecturers.

Within the framework of the CE project, the Institute is engaged in top scientific research, in the training of scientists from abroad, and is offering long-term research positions for Postdoctoral Fellows and renowned scientists from EU countries. It further develops cooperation with laboratories in EU countries and with the support of the European Commission; it organizes meetings with international participation. The CE project is based on 14 work packages, which include also research programs of visitors from abroad and an overview of the prepared international symposia. The goal of the project is to prepare specific recommendations aiming at the reduction of negative consequences of stress, social and nutritional factors on human health. The main focus is on the following tasks:

• to reduce the risk of cardiovascular disease development in persons chronically exposed to stress conditions;
• to mitigate the effect of chronic stress on mental health (treatment of depression, prevention and improvement of anxiety and affective disorders);
• to assist efforts leading to a decrease of negative forms of behavior (drug addiction, alcoholism) related to changes in social relationships;
• to improve diagnostic and therapeutic procedures in patients with hypertension, obesity, and diabetes.

Insight into neuroendocrine and metabolic processes involved in the development of the given disorders is expected to provide an important social and economic contribution.

The financial support received for activities of the Centre of Excellence from the European Commission is primarily assigned for covering expenses associated with the stay of foreign scientists at the Institute, involving also the creation of appropriate conditions for their scientific work, which should markedly enhance the methodological level of research and open up new lines of further cooperation. In the past, our researchers were sent for training to institutes abroad, nowadays scientists from countries of the EU come to work in the laboratories of our Institute.

In agreement with overall objective, the project has provided experimental and clinical data that could help to reduce the negative impact of environmental factors, particularly of chronic stress exposure and inadequate nutrition, on human health. Exaggerated release of stress hormones and negative influences of some nutritional factors or pollutants have been shown to contribute to increased incidence and course of cardiovascular, metabolic, oncological and mental diseases. The main achievements of the project can be divided into three aspects, namely (1) obtaining high quality scientific data with relevance to clinical research, (2) active and broad cooperation among European scientists and (3) significant improvement of the training process.

With the participation of both senior and junior visitors of the Centre from other European laboratories (Sweden, Austria, Hungary, France, Czech Republic, Poland, Greece, Germany), many original data have been obtained contributing to the knowledge on possible reduction of negative impact of environmental factors, particularly of chronic stress exposure and inadequate nutrition, on human health.

Reduction of negative impact of stress exposure. Evidence has been obtained to support further clinical research on the following substances to modulate hormone release and treat the negative impact of demanding stressful situations, anxiety and depression:

antiepileptic drugs, mood stabilizers

• Felbamate and other antiepileptic drugs are recommended for further evaluation in the treatment of affective disorders. We have demonstrated that repeated social conflicts in a mouse model result in stress hormone release. Stress hormone and behavioural changes were reduced by treatment with felbamate, an antiepileptic drug modulating glutamatergic neurotransmission. Data indicate that modulation of stress hormone release contributes to the mood stabilizing and possible antidepressant effects of this drug.

• Drugs influencing glutamate neurotransmission are recommended for further testing in the treatment of drug addiction and non-substance addictive behavior. The recommendation is based on the evidence provided, namely that brain glutamate receptors are involved in the development of compulsory wheel running in rats. In particular, treatment of rats with phenytoin, drug acting also on glutamate release, prevented the development of compulsory running.
• It is recommended to aim future research also to pharmacological modulation of peripheral glutamate receptors. The changes observed at the molecular level in peripheral tissues, such as adrenal gland and liver using animal models of stress and depression, might contribute to high co-morbidity of affective disorders, cardiovascular diseases and cancer.

• Neuroendocrine consequences of medications during pregnancy should be considered. We were the first to demonstrate neuroendocrine changes in adult offspring of mothers treated during gravidity with antiepileptics. Our observations revealed a new component of fetal hydantoin syndrome, namely an enhanced catecholamine response during stress.

• Lamotrigin, an antiepileptic and mood-stabilizing drug is recommended for further clinical consideration. In a double blind, placebo controlled trial, acute administration of lamotrigine, which is known to inhibit glutamate release, modified neuroendocrine and cardiovascular activity during psychosocial stress in healthy men. The modulatory action of lamotrigine on hormone release observed during psychosocial stress is thought to be important for its mood stabilizing effects used in the treatment of bipolar disorder.

antidepressants

• A new aspect of the mechanism of action of antidepressant drugs has been revealed in a placebo controlled double-blind study in healthy volunteers. It has been shown that repeated antidepressant treatment in healthy men does not inhibit, as it is generally believed, but increases neuroendocrine activation during stress. This applies to the action of two antidepressants (citalopram and tianeptine), which have an opposite effect on brain serotonin. It is suggested that an enhancement of the response to acute stress stimuli by antidepressants may be of benefit for patients with depression, in which an attenuated stress response has been reported.

extract of Ginkgo biloba

• We have provided evidence that the standardized extract from ginkgo biloba EGb 761 has an inhibitory action on blood pressure and cortisol responses during stress in healthy humans, which might be useful in avoidance of negative consequences of stress exposure in preventive medicine. The trial was performed as a randomized double blind study in a large group of healthy subjects.

pentoxifylline

• Pentoxifylline, a drug with a good safety profile, is recommended for further clinical research to obtain preventive treatments to ameliorate the negative consequences of daily-life stress situations. The data obtained demonstrate stress-induced damage to vessels (endotheliemia) and its improvement by the treatment with pentoxifylline in rats.

Several new mechanisms involved in the stress response have been revealed, which contribute to better understanding and possible treatment of stress-related disorders:

preclinical studies

·         Findings on gene expression of an adrenaline synthesizing enzyme in cardiac tissues in normal and corticoliberin gene deficient mice contribute to the understanding of regulatory mechanism of heart function by glucocorticoids, especially in stress situations.

• A new mechanism of the sympathoadrenal system regulation during pain exposure has been described. The mechanism involves a pain-induced reduction of adrenaline release most probably via an activation of adrenaline interneurons at the level of preganglionic neurons.
• We have brought evidence that salsolinol, a newly described prolactoliberin, has an inhibitory effect on sympathoadrenal system activity, especially during stress conditions. This finding represents a new regulatory mechanism for the sympathoadrenal system, which may help to explain some pathological changes in the cardiovascular system and it may be a target for future development of prophylactic treatments.

·         We have obtained evidence for a direct biosynthesis of adrenaline in the sympathetic ganglia, which might clarify the mechanism of signal transmission at the ganglionic level and help to explain some pathological processes in sympathetic terminals of various organs of the animal and human body.

clinical studies

• A new approach in the stress research in humans may be the measurement of non-verbal behavior. Using an own modification of a psychosocial stress test based on public speech we were the first to evaluate non-verbal behavior during stress exposure and its modulation by drug treatment.
• In the evaluation of stress coping and associated hormonal changes in healthy humans, their trait anxiety and other psychological variables have to be considered. In contrast to the traditional view, we have shown that subjects with high anxiety exhibit decreased rather than increased neuroendocrine activation during psychosocial stress in comparison with that in individuals with low trait anxiety levels. We suggest that high trait anxiety may be associated with an inability to respond with adequate hormone release to acute stress stimuli.

Other environmental factors, pollutants and nutrition

social environment

• Evidence has been provided for a significant role of social environment on coping with stress and responding to drug treatments. We have shown that rats kept for a long time in an enriched environment respond differently to repeated stress stimuli and drug treatment than do the animals kept under standard conditions. Thus, environmental enrichment modifies adaptation processes and responses to drug treatments, which could have several practical consequences.
• Enriched environment seems to have a beneficial effect on hormonal responses to immune activation, as demonstrated in an animal model of infection.

heavy metal pollutants

• Our results showed diminished number of insulin receptors in rat adipocytes, which could account for diabetogenic effect of long lasting cadmium intake. Ingestion of mercury by mammals induced significant changes in density of retinoic acid receptors in rat liver that might lead consequently to impairment of the retinoid controlled metabolic pathways in the organism.

nutritional factors

·         A negative impact of  fat with respect to fatty acid composition of diet on the whole body insulin sensitivity and glucose utilisation was demonstrated also by the studies using dietary fat rich in long chain saturated fatty acids. On the other hand enrichment of diet with medium chain triglyceride was able to prevent the development of insulin resistance .

• A new aspect of the mechanism of action of leptin has been revealed. Acute in vivo studies have clearly shown important role of leptin in the regulation of fuel sensing mechanism in obesity.

• Dietary supplements by amino acids are recommended for preventive treatment of stress-related anxiety disorders. We have shown that prolong treatment of subjects with relatively high trait anxiety with amino acids lysine and arginine can induce anxiolytic effects and modify hormonal responses during psychosocial stress.

Active and broad cooperation among European scientists

            The senior visitors of the Centre included leading scientists from Sweden, Austria, Hungary and France. Particular achievements have been obtain with the imput of long-term senior visitors who stayed at the Centre. Post-doctoral fellows from Germany, Sweden , Hungary and Austria staying in the CE mainly for 2 years, took part in research devoted to environmental pollutants, lipid metabolism and insulin resistance as well as mechanisms involved in the stress response. Research of the PhD student from Greece has brought new knowledge on addictive behavior, depression and action of some centrally acting drugs in humans.

            Very successful has been the twinning and networking, which allowed to strengthen the existing and create new collaborations among top European laboratories. Mutual visits (e.g. Austria, France) have inoculated new aspects of research and performance of studies, which otherwise would not have been initiated. One of new aspects is related to the peptide adrenomedullin, a potent endothelium vasodilator involved in the control of cardiovascular function. We were first to demonstrate that adrenomedullin levels change in response to diving. Prolong water immersion in healthy men resulted an increase in plasma adrenomedullin concentrations, which might have a preventing effect on pulmonary or whole body vascular leakage. These and several other important findings were the result of twinning between the Laboratory of Pharmacological Neuroendocrinology of this Centre and Institute of Physiology, University of Graz. The twinning of the Laboratory of Neurohumoral Regulation with the research teams from INSERM U-501, Marseille and Department of Physiology, Insbruck has helped to reveal that the stimulation of insulin secretion induced by cell swelling overcomes physiological inhibitory mechanism and exploits novel signaling pathway. This might be of importance for preparation of new generation of drugs to stimulate insulin production. A very interesting finding of the Bratislava - Innsbruck collaboration has been the evidence of up-regulation of specific ion channels gene expression and activity by glucose. The twinning with other successful Centres of Excellence in the Czech Republic and Hungary resulted not only to good publications in international journal, but also to apparently long lasting relationships. A new collaboration between the Laboratory of Pharmacological Neuroendocrinology and a Greek research team has recently started and it led to a common grant application. Moreover, three successful high-level international scientific meetings have been held. These meetings kept or even exceeded international standards, hosted distinguished scientists in the field and strengthened the collaborative efforts. A special meeting has been organized at the very end of project period aimed to disseminate the results obtained at a broad forum of endocrinologists. Another aim was to gather current and former foreign visitors of the Centre together with the scientists to promote discussions on common scientific problems and collaborations. The success and fulfillment of the objectives of this Centre of Excellence project are documented also by the fact that all the visitors expressed their wish to remain in contact with the Centre and continue in collaborative work. Altogether, the research work on this project resulted in 78 papers published mainly in international scientific journals; in 129 abstracts, 30 meeting presentations and selected results have been the subject of patent application.

Improvement of the training process

Both domestic and foreign PhD students and young researchers have profited from the presence of senior visitors of the CE, in particular from Austria, Sweden and Hungary. The education of young scientists and PhD students has been improved by introducing new behavioral models and by course on modern statistical methods. About 20 PhD students and young researchers attended the course and it was very successful.

Improvement of the training process thanks to the project of Centre of Excellence contributed to the high quality of PhD theses of domestic students. All PhD graduates of this Centre exceed the requirements of PhD committees as to the scientific quality and number of publications. It is documented also by the fact that two of our PhD graduates have been evaluated as best graduates of PhD courses at the Slovak Academy of Sciences. Young researchers of the CE have received several awards, e.g. Young Investigator Price of the Ministry of Education of the Slovak Republic, awards for the best publication of the year of medical societies, young investigator grants. International reputation of the CE as a training place increased importantly, e.g. a medical doctor from Austria has recently started an external PhD study in the CE in Slovakia.